Methods of increasing strength and functionality with gdf8 inhibitors

ABSTRACT

The disclosure provides compositions, kits, and methods of using a GDF-8 inhibitor to increase lean muscle mass. In embodiments, a GDF-8 inhibitor is an antibody or antigen binding fragment thereof that specifically binds GDF-8. In embodiments, a method comprises providing an exercise regimen for the subject, and administering a composition comprising an effective amount of a GDF-8 inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. patentapplication Ser. No. 15/099,701, filed Apr. 15, 2016, which claims thebenefit of U.S. Provisional Application No. 62/147,853, filed Apr. 15,2015, and U.S. Provisional Application No. 62/234,899, filed Sep. 30,2015, and U.S. Provisional Application No. 62/261,528, filed Dec. 1,2015. The disclosures of each which are incorporated herein in theirentirety.

INTRODUCTION

A decrease in skeletal muscle mass appears to play a significantpathological role in the progression of a wide variety of disordersassociated with aging, frailty, and certain metabolic conditions. In theelderly, conditions such as sarcopenia and specific events such as a hipfracture may be directly tied to a significant loss of global musclemass. In both older and younger populations, the recovery fromimmobilization and orthopedic surgeries may be linked to the degree ofacute muscle loss associated with both muscle disuse and atrophy drivenby the procedure. In addition, the gain or maintenance of skeletalmuscle mass can result in the prevention of obesity as well as metabolicimprovements.

Myostatin or growth differentiation factor 8 (GDF8) is a soluble TGF-βsuperfamily ligand. It is a negative regulator of muscle growthexpressed principally in skeletal muscle but low expression has beenreported in other tissues such as heart and adipose, at levelsapproximately 100-fold lower than that seen in skeletal muscle(McPherron Nature 387:83, 1997, Sharma J. Cell. Phys. 180:1, 1999, LeeAnnrev. Cell Dev. Biol. 20:61, 2004, Allen Pysiol. Rev. 88:257, 2008,Heineke Cir. 121:419, 2010). Mature myostatin is highly conserved amongspecies, and inactivating mutations of the myostatin gene leads to ahypermuscular phenotype in multiple species including mice, cattle,dogs, and humans. Conversely, overexpression of myostatin in mice (byinjection of transfected CHO cells into thighs of athymic mice orgeneration of striated muscle transgenic mice) caused a significantdecrease in body mass due to decrease in muscle fiber size (McPherron1997cied supra, Grobet Nat. Genet. 17:71, 1997, Mosher PLOS Genet.3:e79, 2007, Schuelke NEJM 350:2682, 2004). While the myostatin nullmouse phenotype demonstrates the importance of myostatin in the controlof muscle size during development, hypertrophy can also be elicited inadult muscle through inhibition of myostatin by neutralizing antibodies,decoy receptors, or other antagonists. However, the effects on myostatininhibitors on cardiac tissue in function may provide an undesirable sideeffect profile for use of these inhibitors to treat conditions likesarcopenia, and metabolic conditions.

Methods of Increasing Strength and Functionality with Gdf8 Inhibitors

This disclosure provides methods and formulations for use in the methodsas described herein. GDF8 inhibitors are useful to enhance lean musclemass in a subject, for example, in combination with exercise. In someembodiments, the subject is a subject that that does not have a diseaseor disorder that significantly limits the subject's ability toparticipate in resistance training. In embodiments, the disease ordisorder is one in which a physician has recommended limited physicalactivity for the subject or in which exercise is contraindicated such asuncontrolled diabetes, recent myocardial infarction, unstable cardiacconditions, acute heart failure, severe myocarditis, uncontrolledhypertension, cardiac valve disease requiring surgery, and severe aorticstenosis.

In embodiments, a method for increasing lean body mass in a subjectcomprises providing an exercise regimen for the subject, andadministering a composition comprising an effective amount of a GDF-8inhibitor wherein, the effective amount is at least 400 mg. Inembodiments, the exercise regimen includes, without limitation,resistance training, weight training, yoga, aerobic exercise, andpilates. In embodiments, a GDF8 inhibitor is an antibody or antigenbinding fragment that specifically binds GDF8. In embodiments, theantibody or antigen binding fragment comprises heavy chain CDRscontained within a heavy chain variable region selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 34, SEQ ID NO: 50,SEQ ID NO: 66, SEQ ID NO: 82, SEQ ID NO: 98, SEQ ID NO: 114, SEQ ID NO:130, SEQ ID NO: 146, SEQ ID NO: 162, SEQ ID NO: 178, SEQ ID NO: 194, SEQID NO: 210, SEQ ID NO: 226, SEQ ID NO: 242, SEQ ID NO: 258, SEQ ID NO:274, SEQ ID NO: 290, SEQ ID NO: 306, SEQ ID NO: 360, and SEQ ID NO: 376.In embodiments, the method further comprises an antibody or antigenbinding fragment that comprises light chain CDRS contained within alight chain variable regions selected from the group consisting of SEQID NOs: SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 42, SEQ ID NO: 58, SEQID NO: 74, SEQ ID NO: 90, SEQ ID NO: 106, SEQ ID NO: 122, SEQ ID NO:138, SEQ ID NO: 154, SEQ ID NO: 170, SEQ ID NO: 186, SEQ ID NO: 202, SEQID NO: 218, SEQ ID NO: 234, SEQ ID NO: 250, SEQ ID NO: 266, SEQ ID NO:282, SEQ ID NO: 298, SEQ ID NO: 314, SEQ ID NO: 322, SEQ ID NO: 368, andSEQ ID NO: 384.

In embodiments, compositions are formulated to contain an effectiveamount of a GDF8 inhibitor to increase lean muscle mass. In embodiments,an effective amount is at least 0.1 mg/kg to about 10 gm/kg, 1 mg/kg toabout 1 gm/kg, or 10 mg/kg to 100 mg/kg. In embodiments, the compositionis administered at least once a week, twice a week, three times a week,four times a week, or five times a week. In embodiments, the exerciseregimen is followed for at least 12 weeks. In embodiments, thecompositions are formulated for intravenous, subcutaneous, or oraladministration.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (A) shows the results of the MAD study designed to assesses thesafety, tolerability, pharmacokinetics (PK), immunogenicity, andpharmacodynamic (PD) effects of REGN1033 (anti-GDF8 antibody)administered subcutaneously (SC) in healthy volunteers 60 years of ageand older. A total of 5 cohorts with 12 subjects enrolled in each cohortwere studied. Subjects received SC doses of REGN1033 (n=9) or placebo(n=14). The planned REGN1033 dose regimens were 100, 200, or 400 mg Q2Wfor a total of 6 doses per subject and 200 mg or 400 mg Q4W for a totalof 3 doses per subject. The % lean body mass was determined using dualenergy x-ray absorptiometry (DEXA). FIG. 1 (B) shows the percent changein lean muscle mass per study visit per group in subjects receivingPlacebo alone (third line from top), Placebo plus resistance training(RT)(bottom line), 400 mg SC of REGN1033 alone, for a total of 6 dosesover the study period (second line from top), and 400 mg SC of REGN1033plus RT (top line), for a total of 6 doses over the study period.

FIG. 2 shows the % change in appendicular fat mass per study visit foreach group in subjects receiving Placebo alone (third line from top),Placebo plus resistance training (RT)(bottom line), 400 mg SC ofREGN1033 alone (second line from top line), for a total of 6 doses overthe study period, and 400 mg SC of REGN1033 plus RT (top line), for atotal of 6 doses over the study period. Placebo alone and 400 mg SC ofREGN1033 alone lines overlap one another.

FIG. 3 (A) shows percent change in thigh muscle mass includingintramuscular fat per study visit per group in subjects receivingPlacebo alone (third line from top), Placebo plus resistance training(RT)(bottom line), 400 mg SC of REGN1033 alone, for a total of 6 dosesover the study period (second line from top), and 400 mg SC of REGN1033plus RT (top line), for a total of 6 doses over the study period. FIG. 3(B) shows percent change in thigh muscle mass excluding intramuscularfat per study visit per group in subjects receiving Placebo alone (thirdline from top), Placebo plus resistance training (RT) (bottom line), 400mg SC of REGN1033 alone, for a total of 6 doses over the study period(second line from top), and 400 mg SC of REGN1033 plus RT (top line),for a total of 6 doses over the study period. Placebo alone and Placeboplus RT lines overlap one another.

FIG. 4 shows percent change in gynoid fat per study visit per group insubjects receiving Placebo alone (top line), Placebo plus resistancetraining (RT)(second line from top), 400 mg SC of REGN1033 alone, for atotal of 6 doses over the study period (third line from top), and 400 mgSC of REGN1033 plus RT, for a total of 6 doses over the study period(bottom line). Placebo plus RT and 400 mg SC of REGN1033 alone linesoverlap one another.

FIG. 5 shows percent change in chest press per study visit per group insubjects receiving Placebo alone (bottom line), Placebo plus resistancetraining (RT)(third line from top), 400 mg SC of REGN1033 alone, for atotal of 6 doses over the study period (second line from top), and 400mg SC of REGN1033 plus RT (top line), for a total of 6 doses over thestudy period.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis disclosure is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the invention will be limited only bythe appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used herein, the term “about,” when used in reference to a particularrecited numerical value, means that the value may vary from the recitedvalue by no more than 1%. For example, as used herein, the expression“about 100” includes 99 and 101 and all values in between (e.g., 99.1,99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of theinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

“Human Growth Differentiation Factor-8”, “GDF8” and “myostatin” are usedinterchangeably to refer to the protein encoded by the nucleic acidsequence of SEQ ID NO: 338 and the protein having the amino acidsequence of SEQ ID NO: 339 (propeptide) and SEQ ID NO: 340 (matureprotein).

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In different embodiments of the invention, the FRs of theanti-GDF8 antibody (or antigen-binding portion thereof) may be identicalto the human germline sequences, or may be naturally or artificiallymodified. An amino acid consensus sequence may be defined based on aside-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)₂ fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR)). Other engineered molecules,such as diabodies, triabodies, tetrabodies and minibodies, are alsoencompassed within the expression “antigen-binding fragment,” as usedherein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a VH domain associated with a V_(L)domain, the V_(H) and V_(L) domains may be situated relative to oneanother in any suitable arrangement. For example, the variable regionmay be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) or V_(L)-V_(L)dimers. Alternatively, the antigen-binding fragment of an antibody maycontain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)—C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the invention may comprise ahomo-dimer or hetero-dimer (or other multimer) of any of the variableand constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric VH or VLdomain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe invention using routine techniques available in the art.

The antibodies of the invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656(1998)).

The term “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Specific binding can becharacterized by a dissociation constant of 1×10⁻⁶ M or less. Methodsfor determining whether two molecules specifically bind are well knownin the art and include, for example, equilibrium dialysis, surfaceplasmon resonance, and the like. For example, an antibody that“specifically binds” human GDF8, as used in the context of theinvention, includes antibodies that bind human GDF8 or portion thereof(e.g., a peptide comprising at least 6 contiguous amino acids of SEQ IDNO:340) with a K_(D) of less than about 1000 nM, less than about 500 nM,less than about 300 nM, less than about 200 nM, less than about 100 nM,less than about 90 nM, less than about 80 nM, less than about 70 nM,less than about 60 nM, less than about 50 nM, less than about 40 nM,less than about 30 nM, less than about 20 nM, less than about 10 nM,less than about 5 nM, less than about 4 nM, less than about 3 nM, lessthan about 2 nM, less than about 1 nM or less than about 0.5 nM, asmeasured in a surface plasmon resonance assay. (See, e.g., Example 3,herein). An isolated antibody that specifically binds human GDF8 may,however, have cross-reactivity to other antigens, such as GDF8 moleculesfrom other species.

The term “high affinity” antibody refers to those antibodies capable ofbinding to GDF8 with a dissociation constant (K_(D)) of about 10⁻⁸ M orless, about 10⁻⁹M or less, about 10⁻¹⁰ M or less, about 10⁻¹¹ M or less,or about 10⁻¹² M or less, as measured by surface plasmon resonance,e.g., BIACORE™ or solution-affinity ELISA.

By the term “slow off rate” or “Koff” is meant an antibody thatdissociates from GDF8 with a rate constant of 1×10⁻³ s⁻¹ or less,preferably 1×10⁻⁴ s⁻¹ or less, as determined by surface plasmonresonance, e.g., BIACORE™.

A “neutralizing” or “blocking” antibody, is intended to refer to anantibody whose binding to GDF8 results in inhibition of the biologicalactivity of GDF8. This inhibition of the biological activity of GDF8 canbe assessed by measuring one or more indicators of GDF8 biologicalactivity. These indicators of GDF8 biological activity can be assessedby one or more of several standard in vitro or in vivo assays known inthe art.

As used herein, the expression “anti-GDF8 antibody” also includesmultispecific antigen-binding molecules (e.g., bispecific antibodies)wherein at least one binding domain (e.g., “binding arm”) of themultispecific antigen-binding molecule specifically binds GDF8.

Exemplary anti-GDF8 antibodies that can be used in the context of theinvention include, e.g., the fully-human anti-GDF8 antibody H4H1657N2(Regeneron/Sanofi) (e.g., an anti-GDF8 antibody comprising the heavy andlight chain variable regions having amino acid sequences SEQ ID NO: 360and SEQ ID NO: 368, respectively, as set forth in U.S. Pat. No.8,840,894). Other GDF8 antagonists that can be used in the context ofthe methods of the invention include anti-GDF8 antibodies (e.g., theantibody designated 2_112_1 (ATCC deposit designation PTA-6574)) as setforth in U.S. Pat. No. 7,807,159, anti-GDF8 antibodies (e.g. 12A5-5) asset forth in U.S. Pat. No. 8,999,343 and US Publication No.2013/0209489, anti-GDF8 antibodies (e.g., 10B3H8L5 and10B3H8L5-Fc-disabled) as set forth in US Publication No. 2013/0142788,the anti-GDF8 antibody stamulumab/MYO-29 as set forth in, e.g., U.S.Pat. No. 8,940,874, anti-GDF8 antibodies (e.g., RK22/PF-0625616) as setforth in U.S. Pat. No. 8,415,459, anti-GDF8 antibodies (e.g., JA-16) asset forth in U.S. Pat. No. 7,731,961, anti-GDF8 antibodies (e.g., RK35)as set forth in U.S. Pat. No. 8,496,934, anti-GDF8 antibodies (e.g.,OGD1.0.0) as set forth in U.S. Pat. No. 8,992,913, anti-GDF8 Fabmolecules as set forth in European Patent No. 1 773 041 B1, anti-GDF8antibodies (e.g., 41C1E4) as set forth in U.S. Pat. No. 7,632,499, andanti-GDF8 antibodies (e.g., C12, C12-N93H and/or 510C2) as set forth in,e.g., U.S. Pat. Nos. 7,635,760 and 8,063,188. The disclosures of all ofthe aforementioned patents and patent application publications areincorporated by reference herein in their entireties

The fully-human anti-GDF8 antibodies disclosed herein may comprise oneor more amino acid substitutions, insertions and/or deletions in theframework and/or CDR regions of the heavy and light chain variabledomains as compared to the corresponding germline sequences. Suchmutations can be readily ascertained by comparing the amino acidsequences disclosed herein to germline sequences available from, forexample, public antibody sequence databases. The invention includesantibodies, and antigen-binding fragments thereof, which are derivedfrom any of the amino acid sequences disclosed herein, wherein one ormore amino acids within one or more framework and/or CDR regions areback-mutated to the corresponding germline residue(s) or to aconservative amino acid substitution (natural or non-natural) of thecorresponding germline residue(s) (such sequence changes are referred toherein as “germline back-mutations”). A person of ordinary skill in theart, starting with the heavy and light chain variable region sequencesdisclosed herein, can easily produce numerous antibodies andantigen-binding fragments which comprise one or more individual germlineback-mutations or combinations thereof. In certain embodiments, all ofthe framework and/or CDR residues within the V_(H) and/or V_(L) domainsare mutated back to the germline sequence. In other embodiments, onlycertain residues are mutated back to the germline sequence, e.g., onlythe mutated residues found within the first 8 amino acids of FR1 orwithin the last 8 amino acids of FR4, or only the mutated residues foundwithin CDR1, CDR2 or CDR3. Furthermore, the antibodies of the inventionmay contain any combination of two or more germline back-mutationswithin the framework and/or CDR regions, i.e., wherein certainindividual residues are mutated back to the germline sequence whilecertain other residues that differ from the germline sequence aremaintained. Once obtained, antibodies and antigen-binding fragments thatcontain one or more germline back-mutations can be easily tested for oneor more desired property such as, improved binding specificity,increased binding affinity, improved or enhanced antagonistic oragonistic biological properties (as the case may be), reducedimmunogenicity, etc. Antibodies and antigen-binding fragments obtainedin this general manner are encompassed within the invention.

The invention also includes anti-GDF8 antibodies comprising variants ofany of the HCVR, LCVR, and/or CDR amino acid sequences disclosed hereinhaving one or more conservative substitutions. For example, theinvention includes anti-GDF8 antibodies having HCVR, LCVR, and/or CDRamino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4or fewer, etc. conservative amino acid substitutions relative to any ofthe HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. In oneembodiment, the antibody comprises an HCVR having an amino acid sequenceselected from SEQ ID NO:360 and 376 with 8 or fewer conservative aminoacid substitutions. In another embodiment, the antibody comprises anHCVR having an amino acid sequence selected from SEQ ID NO:360 and 376with 6 or fewer conservative amino acid substitutions. In anotherembodiment, the antibody comprises an HCVR having an amino acid sequenceselected from SEQ ID NO:360 and 376 with 4 or fewer conservative aminoacid substitutions. In another embodiment, the antibody comprises anHCVR having an amino acid sequence selected from SEQ ID NO:360 and 376with 2 or fewer conservative amino acid substitutions. In oneembodiment, the antibody comprises an LCVR having an amino acid sequenceselected from SEQ ID NO:368 and 384 with 8 or fewer conservative aminoacid substitutions. In another embodiment, the antibody comprises anLCVR having an amino acid sequence selected from SEQ ID NO:368 and 384with 6 or fewer conservative amino acid substitutions. In anotherembodiment, the antibody comprises an LCVR having an amino acid sequenceselected from SEQ ID NO:368 and 384 with 4 or fewer conservative aminoacid substitutions. In another embodiment, the antibody comprises anLCVR having an amino acid sequence selected from SEQ ID NO:368 and 384with 2 or fewer conservative amino acid substitutions.

In certain embodiments, antibody or antibody fragment of the inventionmay be conjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin, a chemotherapeutic drug, and immunosuppressant or aradioisotope.

An “isolated antibody,” as used herein, means an antibody that has beenidentified and separated and/or recovered from at least one component ofits natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, tissueor cell in which the antibody naturally exists or is naturally producedis an “isolated antibody” for purposes of the invention. An isolatedantibody also includes an antibody in situ within a recombinant cell, aswell as an antibody that has been subjected to at least one purificationor isolation step. According to certain embodiments, an isolatedantibody may be substantially free of other cellular material and/orchemicals.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. For example, an antibody is said tospecifically bind an antigen when the K_(D) is less than or equal to10⁻⁸ M, less than or equal to 10⁻⁹ M, or less than or equal to 10⁻¹⁰ M.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60, 70%, 80% or 90% w/w ofa protein sample, usually about 95%, and preferably over 99% pure.Protein purity or homogeneity may be indicated by a number of means wellknown in the art, such as polyacrylamide gel electrophoresis of aprotein sample, followed by visualizing a single polypeptide band uponstaining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

The term “polypeptide analog or variant” as used herein refers to apolypeptide that is comprised of a segment of at least 25 amino acidsthat has substantial identity to a portion of an amino acid sequence andthat has at least one of the following properties: (1) specific bindingto GDF8 under suitable binding conditions, or (2) ability to block thebiological activity of GDF8. Typically, polypeptide analogs or variantscomprise a conservative amino acid substitution (or insertion ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, at least 50, 60, 70, 80, 90,100, 150 or 200 amino acids long or longer, and can often be as long asa full-length naturally-occurring polypeptide.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmutations of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton 1984 W. H. Freeman andCompany, New York; Introduction to Protein Structure (Branden & Tooze,eds., 1991, Garland Publishing, NY); and Thornton et at. 1991 Nature354:105, which are each incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry asdrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (see, for example, Fauchere (1986) J. Adv. Drug Res.15:29; and Evans et al. (1987) J. Med. Chem. 30:1229, which areincorporated herein by reference. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may also be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo et al.(1992) Ann. Rev. Biochem. 61:387, incorporated herein by reference), forexample, by adding internal cysteine residues capable of formingintramolecular disulfide bridges which cyclize the peptide.

As applied to polypeptides, the term “substantial identity” or“substantially identical” means that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least about 80% sequence identity, at least about90%, at least about 95%, at least about 98% or at least about 99%sequence identity. Preferably, residue positions which are not identicaldiffer by conservative amino acid substitutions. A “conservative aminoacid substitution” is one in which an amino acid residue is substitutedby another amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of similarity may be adjustedupwards to correct for the conservative nature of the substitution.Means for making this adjustment are well-known to those of skill in theart. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, hereinincorporated by reference. Examples of groups of amino acids that haveside chains with similar chemical properties include 1) aliphatic sidechains: glycine, alanine, valine, leucine and isoleucine; 2)aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; and 6) sulfur-containing side chainsare cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256:1443-45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000), supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially blastp or tblastn, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389 402, each of which is hereinincorporated by reference.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, at least about 20 residues, atleast about 24 residues, at least about 28 residues, or at least about35 residues. When searching a database containing sequences from a largenumber of different organisms, it is preferable to compare amino acidsequences.

The term “effective amount” is a concentration or amount of an antibodyor antigen-binding fragment of an antibody which results in achieving aparticular stated purpose. An “effective amount” of an anti-GDF8antibody or antigen-binding fragment of an antibody thereof may bedetermined empirically. Furthermore, a “therapeutically effectiveamount” is a concentration or amount of an anti-GDF8 antibody orantigen-binding fragment thereof which is effective for achieving astated therapeutic effect. This amount may also be determinedempirically.

As used herein, a “healthy subject” refers to a subject that does nothave a disease or disorder that significantly limits the subject'sability to participate in resistance training. In embodiments, thedisease or disorder is one in which a physician has recommended limitedphysical activity for the subject or in which exercise iscontraindicated such as uncontrolled diabetes, recent myocardialinfarction, unstable cardiac conditions, acute heart failure, severemyocarditis, uncontrolled hypertension, cardiac valve disease requiringsurgery, and severe aortic stenosis.

As used herein, “resistance training” refers to a set of exercises thatcauses a muscle to contract against an external resistance. The externalresistance includes for example, a weight, a band, a kettleball, or thesubject's body weight.

As used herein, “exercise regimen” refers to a plan of exercises. Inembodiments, an exercise regimen includes exercises, such as, resistancetraining, weigh training, aerobic training, walking, interval training,yoga, and combinations thereof.

Aspects of the Disclosure

The disclosure provides compositions, kits, and methods of using GDF-8inhibitors to increase lean body mass. In embodiments, the GDF-8inhibitor is an antibody or antigen binding fragment thereof thatspecifically binds GDF-8.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the invention to make human antibodies thatspecifically bind to GDF8.

Using VELOCIMMUNE™ technology or any other known method for generatingmonoclonal antibodies, high affinity chimeric antibodies to GDF8 areinitially isolated having a human variable region and a mouse constantregion. As in the experimental section below, the antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, etc. The mouse constant regions arereplaced with a desired human constant region to generate the fullyhuman antibody of the invention, for example wild-type or modified IgG1or IgG4. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

In general, the antibodies of the instant invention possess very highaffinities, typically possessing K_(D) of from about 10⁻¹² through about10⁻⁹ M, when measured by binding to antigen either immobilized on solidphase or in solution phase. The mouse constant regions are replaced withdesired human constant regions to generate the fully human antibodies ofthe invention, for example wild-type IgG1 (SEQ ID NO: 335) or IgG4 (SEQID NO: 336), or modified IgG1 or IgG4 (for example, SEQ ID NO: 337).While the constant region selected may vary according to specific use,high affinity antigen-binding and target specificity characteristicsreside in the variable region.

Antibodies or Antigen Binding Fragments Specific for GDF-8

The invention includes anti-GDF8 antibodies and antigen-bindingfragments of antibodies which bind specific epitopes of human GDF8 (SEQID NO:340) and are capable of blocking the biological activity of GDF8.In one embodiment, the antibody or antigen-binding fragment thereofbinds within an epitope comprising amino acids residues 1 to 109; 1 to54; 1 to 44; 1 to 34; 1 to 24; and 1 to 14. In another embodiment, theantibody or antigen-binding fragment thereof binds within an epitopecomprising of amino acid residues 65 to 72; 35 to 109; 45 to 109; 55 to109; 65 to 109; 75 to 109; 85 to 109; 92 to 109; or 95 to 109. Inanother embodiment, the antibody or antigen-binding fragment thereofbinds within an epitope comprising amino acid residue 48 to 72; 48 to69; 48 to 65; 52 to 72; 52 to 65; or 56 to 65. In specific embodiments,the antibody or antigen-binding fragment thereof may bind within 2 ormore epitopes.

The invention also includes antibodies and antigen-binding fragmentsthereof that bind wild-type mature GDF8 (SEQ ID NO: 340) but do not bindisolated peptides having less than the full amino acid sequence of SEQID NO: 340. For example, the invention includes anti-GDF8 antibodiesthat bind wild-type mature GDF8 (SEQ ID NO: 340) but do not bindisolated peptides consisting of 10 to 40 contiguous amino acids of SEQID NO: 340. The invention also includes anti-GDF8 antibodies that do notbind any linear epitopes within wild-type mature GDF8. In certainembodiments of the invention, the anti-GDF8 antibodies bind wild-typemature human GDF8 comprising SEQ ID NO: 340 but do not bind one or moreisolated GDF8 peptides having an amino acid sequence selected from thegroup consisting of amino acids 1-14, 1-18, 17-42, 48-65, 48-69, 48-72,52-65, 52-72, 56-65, 56-72, 65-72, 73-90, 75-105 and 91-105, of SEQ IDNO:340. In certain embodiments, the anti-GDF8 antibodies do not bind anyof the aforementioned GDF8 peptides. Methods for determining whether agiven antibody is able to bind a particular GDF8 peptide are known topersons of ordinary skill in the art.

The invention also includes isolated human antibodies, orantigen-binding fragments thereof, that specifically bind to wild-typemature human GDF8 (e.g., a protein or polypeptide comprising SEQ ID NO:340), but do not bind to a chimeric GDF8 construct in which certainamino acids of GDF8 are replaced with the corresponding amino acidsequence(s) from a non-identical but related protein such as TGFβ-1. Inone example, the chimeric construct is a GDF8/TGFβ-1 chimera in whichamino acids 48-72 of mature GDF8 are replaced with the correspondingamino acid sequence of TGFβ-1 (e.g., amino acids 49-76 of TGFβ-1). Anexample of one such chimera is represented by SEQ ID NO: 352. Thus, incertain embodiments, the antibodies of the invention specifically bindto wild-type mature human GDF8 (SEQ ID NO: 340) but do not bind to thechimeric GDF8/TGFβ-1 construct of SEQ ID NO: 352, indicating that theepitope to which such antibodies bind includes or encompasses aminoacids located within residues 48 to 72 of SEQ ID NO: 340. Blockingbioassays can also be used to indirectly ascertain if an antibody bindswild-type mature human GDF8 (SEQ ID NO: 340) and does not bind achimeric GDF8/TGFβ-1 construct, e.g., the construct of SEQ ID NO: 352.For example, an antibody which blocks the bioactivity of wild-typemature human GDF8 but does not block the bioactivity of a chimericGDF8/TGFβ-1 is deemed to bind to the portion of GDF8 that is replaced bythe corresponding TGFβ-1 sequence in the chimeric construct.

Similarly, the invention also includes isolated human antibodies, orantigen-binding fragments thereof, that block wild-type matureGDF8-mediated activity in a bioassay but do not block activity of achimeric GDF8 construct (e.g., a GDF8/TGFβ-1 chimera in which aminoacids 48-72 of mature GDF8 are replaced with the corresponding aminoacid sequence of TGF3-1 (e.g., amino acids 49-76 of TGFβ-1).

The invention includes anti-GDF8 antibodies that bind to the sameepitope as any of the specific exemplary antibodies described herein.Likewise, the invention also includes anti-GDF8 antibodies thatcross-compete for binding to GDF8 or a GDF8 fragment with any of thespecific exemplary antibodies described herein.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-GDF8 antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-GDF8antibody of the invention, the reference antibody is allowed to bind toa GDF8 protein or peptide under saturating conditions. Next, the abilityof a test antibody to bind to the GDF8 molecule is assessed. If the testantibody is able to bind to GDF8 following saturation binding with thereference anti-GDF8 antibody, it can be concluded that the test antibodybinds to a different epitope than the reference anti-GDF8 antibody. Onthe other hand, if the test antibody is not able to bind to the GDF8molecule following saturation binding with the reference anti-GDF8antibody, then the test antibody may bind to the same epitope as theepitope bound by the reference anti-GDF8 antibody of the invention.Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, Biacore, flow cytometryor any other quantitative or qualitative antibody-binding assayavailable in the art. In accordance with certain embodiments of theinvention, two antibodies bind to the same (or overlapping) epitope if,e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are deemedto bind to the same epitope if essentially all amino acid mutations inthe antigen that reduce or eliminate binding of one antibody reduce oreliminate binding of the other. Two antibodies are deemed to have“overlapping epitopes” if only a subset of the amino acid mutations thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other.

To determine if an antibody competes for binding with a referenceanti-GDF8 antibody, the above-described binding methodology is performedin two orientations: In a first orientation, the reference antibody isallowed to bind to a GDF8 molecule under saturating conditions followedby assessment of binding of the test antibody to the GDF8 molecule. In asecond orientation, the test antibody is allowed to bind to a GDF8molecule under saturating conditions followed by assessment of bindingof the reference antibody to the GDF8 molecule. If, in bothorientations, only the first (saturating) antibody is capable of bindingto the GDF8 molecule, then it is concluded that the test antibody andthe reference antibody compete for binding to GDF8. As will beappreciated by a person of ordinary skill in the art, an antibody thatcompetes for binding with a reference antibody may not necessarily bindto the same epitope as the reference antibody, but may sterically blockbinding of the reference antibody by binding an overlapping or adjacentepitope.

The invention provides human or humanized antibodies and antigen-bindingfragments of human or humanized antibodies that specifically bind humangrowth and differentiation factor 8 (GDF8). These antibodies arecharacterized by binding to GDF8 with high affinity and by the abilityto neutralize GDF8 activity. The antibodies can be full-length (forexample, an IgG1 or IgG4 antibody) or may comprise only anantigen-binding portion (for example, a Fab, F(ab′)₂ or scFv fragment),and may be modified to affect functionality, e.g., to eliminate residualeffector functions (Reddy et al. (2000) J. Immunol. 164:1925-1933).

In one embodiment, the antibody of the invention comprises a heavy chainvariable region (HCVR) amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 34, SEQ ID NO: 50,SEQ ID NO: 66, SEQ ID NO: 82, SEQ ID NO: 98, SEQ ID NO: 114, SEQ ID NO:130, SEQ ID NO: 146, SEQ ID NO: 162, SEQ ID NO: 178, SEQ ID NO: 194, SEQID NO: 210, SEQ ID NO: 226, SEQ ID NO: 242, SEQ ID NO: 258, SEQ ID NO:274, SEQ ID NO: 290, SEQ ID NO: 306, SEQ ID NO: 360, and SEQ ID NO: 376,or a substantially identical sequence thereof.

In one embodiment, the antibody of the invention comprises a light chainvariable region (LCVR) amino acid sequence selected from the groupconsisting of SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 42, SEQ ID NO:58, SEQ ID NO: 74, SEQ ID NO: 90, SEQ ID NO: 106, SEQ ID NO: 122, SEQ IDNO: 138, SEQ ID NO: 154, SEQ ID NO: 170, SEQ ID NO: 186, SEQ ID NO: 202,SEQ ID NO: 218, SEQ ID NO: 234, SEQ ID NO: 250, SEQ ID NO: 266, SEQ IDNO: 282, SEQ ID NO: 298, SEQ ID NO: 314, SEQ ID NO: 322, SEQ ID NO: 368,and SEQ ID NO: 384 or a substantially identical sequence thereof.

In one embodiment, the antibody of the invention comprises a HCVR aminoacid sequence and a LCVR amino acid sequence, wherein the HCVR/LCVR pairsequences are selected from the group consisting of SEQ ID NO: 2/10, SEQID NO: 18/26, SEQ ID NO: 34/42, SEQ ID NO: 50/58, SEQ ID NO: 66/74, SEQID NO: 82/90, SEQ ID NO: 98/106, SEQ ID NO: 114/122, SEQ ID NO: 130/138,SEQ ID NO: 146/154, SEQ ID NO: 162/170, SEQ ID NO: 178/186, SEQ ID NO:194/202, SEQ ID NO: 210/218, SEQ ID NO: 226/234, SEQ ID NO: 242/250, SEQID NO: 258/266, SEQ ID NO: 274/282, SEQ ID NO: 290/298, SEQ ID NO:306/314, SEQ ID NO: 114/322, SEQ ID NO: 360/368, and SEQ ID NO: 376/384.

The invention also features a human or humanized antibody orantigen-binding fragment of an antibody comprising a heavy chaincomplementarity determining region 3 (HCDR3) amino acid sequence and alight chain CDR3 amino acid sequence (LCDR3), wherein the HCDR3 aminoacid sequence is selected from the group consisting of SEQ ID NO: 8, SEQID NO: 24, SEQ ID NO: 40, SEQ ID NO: 56, SEQ ID NO: 72, SEQ ID NO: 88,SEQ ID NO: 104, SEQ ID NO: 120, SEQ ID NO: 136, SEQ ID NO: 152, SEQ IDNO: 168, SEQ ID NO: 184, SEQ ID NO: 200, SEQ ID NO: 216, SEQ ID NO: 232,SEQ ID NO: 248, SEQ ID NO: 264, SEQ ID NO: 280, SEQ ID NO: 296, SEQ IDNO: 312, SEQ ID NO: 366, and SEQ ID NO: 382, or a substantiallyidentical sequence thereof, and the LCDR3 amino acid sequence isselected from the group consisting of SEQ ID NO:16, SEQ ID NO: 32, SEQID NO: 48, SEQ ID NO: 64, SEQ ID NO: 80, SEQ ID NO: 96, SEQ ID NO: 112,SEQ ID NO: 128, SEQ ID NO: 144, SEQ ID NO: 160, SEQ ID NO: 176, SEQ IDNO: 192, SEQ ID NO: 208, SEQ ID NO: 224, SEQ ID NO: 240, SEQ ID NO: 256,SEQ ID NO: 272, SEQ ID NO: 288, SEQ ID NO: 304, SEQ ID NO: 320, SEQ IDNO: 328, SEQ ID NO: 374, and SEQ ID NO: 390, or a substantiallyidentical sequence thereof. In another embodiment, the antibody orfragment thereof comprises an HCDR3/LCDR3 amino acid sequence pairselected from the group consisting of SEQ ID NO: 8/16, SEQ ID NO: 24/32,SEQ ID NO: 40/48, SEQ ID NO: 56/64, SEQ ID NO: 72/80, SEQ ID NO: 88/96,SEQ ID NO: 104/112, SEQ ID NO: 120/128, SEQ ID NO: 136/144, SEQ ID NO:152/160, SEQ ID NO: 168/176, SEQ ID NO: 184/192, SEQ ID NO: 200/208, SEQID NO: 216/224, SEQ ID NO: 232/240, SEQ ID NO: 248/256, SEQ ID NO:264/272, SEQ ID NO: 280/288, SEQ ID NO: 296/304, SEQ ID NO: 312/320, SEQID NO: 120/328, SEQ ID NO: 366/374, and SEQ ID NO: 382/390.

In a related embodiment, the antibody or fragment thereof furthercomprises heavy chain CDR1 (HCDR1) and CDR2 (HCDR2) amino acid sequencesand light chain CDR1 (LCDR1) and CDR2 (LCDR2) amino acid sequences,wherein the HCDR1 amino acid sequence is selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 36, SEQ ID NO: 52,SEQ ID NO: 68, SEQ ID NO: 84, SEQ ID NO: 100, SEQ ID NO: 116, SEQ ID NO:132, SEQ ID NO: 148, SEQ ID NO: 164, SEQ ID NO: 180, SEQ ID NO: 196, SEQID NO: 212, SEQ ID NO: 228, SEQ ID NO: 244, SEQ ID NO: 260, SEQ ID NO:276, SEQ ID NO: 292, SEQ ID NO: 308, SEQ ID NO: 362, and SEQ ID NO: 378,or a substantially identical sequence thereof, the HCDR2 amino acidsequence is selected from the group consisting of SEQ ID NO: 6, SEQ IDNO: 22, SEQ ID NO: 38, SEQ ID NO: 54, SEQ ID NO: 70, SEQ ID NO: 86, SEQID NO: 102, SEQ ID NO: 118, SEQ ID NO: 134, SEQ ID NO: 150, SEQ ID NO:166, SEQ ID NO: 182, SEQ ID NO: 198, SEQ ID NO: 214, SEQ ID NO: 230, SEQID NO: 246, SEQ ID NO: 262, SEQ ID NO: 278, SEQ ID NO: 294, SEQ ID NO:310, SEQ ID NO: 364, and SEQ ID NO: 380, or a substantially identicalsequence thereof, the LCDR1 amino acid sequence is selected from thegroup consisting of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ IDNO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124,SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, SEQ IDNO: 204, SEQ ID NO: 220, SEQ ID NO: 236, SEQ ID NO: 252, SEQ ID NO: 268,SEQ ID NO: 284, SEQ ID NO: 300, SEQ ID NO: 316, SEQ ID NO: 324, SEQ IDNO: 370, and SEQ ID NO: 386 or a substantially identical sequencethereof, and the LCDR2 amino acid sequence is selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 30, SEQ ID NO: 46, SEQ ID NO:62, SEQ ID NO: 78, SEQ ID NO: 94, SEQ ID NO: 110, SEQ ID NO: 126, SEQ IDNO: 142, SEQ ID NO: 158, SEQ ID NO: 174, SEQ ID NO: 190, SEQ ID NO: 206,SEQ ID NO: 222, SEQ ID NO: 238, SEQ ID NO: 254, SEQ ID NO: 270, SEQ IDNO: 286, SEQ ID NO: 302, SEQ ID NO: 318, SEQ ID NO: 326, SEQ ID NO: 372,and SEQ ID NO: 388 or a substantially identical sequence thereof. Inanother embodiment, the HCDR1, HCDR2 and HCDR3 are selected from thegroup consisting of SEQ ID NO: 36/38/40, SEQ ID NO: 116/118/120, SEQ IDNO: 228/230/232, SEQ ID NO: 362/364/366, and SEQ ID NO: 378/380/382; andLCDR1, LCDR2 and LCDR3 are selected from the group consisting of SEQ IDNO: 44/46/48, SEQ ID NO: 124/126/128, SEQ ID NO: 236/238/240, SEQ ID NO:370/372/374, and SEQ ID NO: 386/388/390. In yet another embodiment, theheavy and light chain CDRs are selected from the group consisting of SEQID NO: 36/38/40/44/46/48 (e.g. 21-E5), SEQ ID NO:116/118/120/124/126/128 (e.g. 8D12), SEQ ID NO: 228/230/232/236/238/240(e.g. 1A2), SEQ ID NO: 362/364/366/370/372/374 (e.g. H4H1657N2), and SEQID NO: 378/380/382/386/388/390 (e.g. H4H1669P).

In a related embodiment, the invention includes an antibody orantigen-binding fragment of an antibody which specifically binds GDF8,wherein the antibody or fragment comprises the heavy and light chain CDRdomains contained within heavy and light chain variable domain sequencesselected from the group consisting of SEQ ID NO: 2/10, SEQ ID NO: 18/26,SEQ ID NO: 34/42, SEQ ID NO: 50/58, SEQ ID NO: 66/74, SEQ ID NO: 82/90,SEQ ID NO: 98/106, SEQ ID NO: 114/122, SEQ ID NO: 130/138, SEQ ID NO:146/154, SEQ ID NO: 162/170, SEQ ID NO: 178/186, SEQ ID NO: 194/202, SEQID NO: 210/218, SEQ ID NO: 226/234, SEQ ID NO: 242/250, SEQ ID NO:258/266, SEQ ID NO: 274/282, SEQ ID NO: 290/298, SEQ ID NO: 306/314, SEQID NO: 114/322, SEQ ID NO: 360/368, and SEQ ID NO: 376/384.

Methods and techniques for identifying CDRs within HCVR and LCVR aminoacid sequences are well known in the art and can be used to identifyCDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

The invention also provides nucleic acid molecules encoding theantibodies or antigen-binding fragments of the invention. Recombinantexpression vectors carrying the antibody-encoding nucleic acids of theinvention, and host cells into which such vectors have been introduced,are also encompassed by the invention, as are methods of making theantibodies of the invention by culturing the host cells of theinvention.

In one embodiment, the antibody of the invention comprises CDRScontained within or a HCVR encoded by a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 33,SEQ ID NO: 49, SEQ ID NO: 65, SEQ ID NO: 81, SEQ ID NO: 97, SEQ ID NO:113, SEQ ID NO: 129, SEQ ID NO: 145, SEQ ID NO: 161, SEQ ID NO: 177, SEQID NO: 193, SEQ ID NO: 209, SEQ ID NO: 225, SEQ ID NO: 241, SEQ ID NO:257, SEQ ID NO: 273, SEQ ID NO: 289, SEQ ID NO: 305, SEQ ID NO: 359, andSEQ ID NO: 375, or a substantially similar sequence having at least 95%homology thereof.

In one embodiment, the antibody of the invention comprises CDRScontained within or a LCVR encoded by a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 41,SEQ ID NO: 57, SEQ ID NO: 73, SEQ ID NO: 89, SEQ ID NO: 105, SEQ ID NO:121, SEQ ID NO: 137, SEQ ID NO: 153, SEQ ID NO: 169, SEQ ID NO: 185, SEQID NO: 201, SEQ ID NO: 217, SEQ ID NO: 233, SEQ ID NO: 249, SEQ ID NO:265, SEQ ID NO: 281, SEQ ID NO: 297, SEQ ID NO: 313, SEQ ID NO: 321, SEQID NO: 367, and SEQ ID NO: 383 or a substantially similar sequencehaving at least 95% homology thereof.

The invention also features a fully human or humanized antibody orantibody fragment which binds GDF8 with an affinity (expressed as adissociation constant, “K_(D)”) of about 1 nM or less, as measured bysurface plasmon resonance assay (for example, BIACORE™). In certainembodiments, the antibody of the invention exhibits a KDof about 700 pMor less; about 500 pM or less; about 320 pM or less; about 160 pM orless; about 100 pM or less; about 50 pM or less; about 10 pM or less; orabout 5 pM or less.

In one embodiment, the invention provides a fully human or humanizedmonoclonal antibody (mAb) which specifically binds and inhibits humanGDF8 and exhibits an IC₅₀ of less than or equal to about 10 nM; about 5nM or less; about 3 nM or less; about 2 nM or less; about 1 nM or less;about 500 pM or less; or about 200 pM or less, as measured by GDF8inducible luciferase assay. As shown in the experimental section below,some of the anti-GDF8 antibodies of the invention block the activity ofclosely related proteins, such as GDF11, with a much higher IC₅₀ thanGDF8 in a luciferase bioassay. In one embodiment, the invention providesan antibody or antigen-binding fragment of an antibody that exhibits atleast about 10-fold, at least about 50-fold, at least about 100-fold, atleast about 200-fold, at least about 500-fold, at least about 1000-fold,or at least about 1500-fold higher IC₅₀ for blocking GDF11 activityrelative to GDF8.

The invention encompasses anti-GDF8 antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof a galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

The invention includes anti-GDF8 antibodies which bind specific epitopesof GDF8 and are capable of blocking the biological activity of GDF8. Ina first embodiment, the antibody of the invention binds an epitope ofthe mature GDF8 protein (SEQ ID NO:340) within amino acids from about 1to about 109; from about 1 to about 54; from about 1 to about 44; fromabout 1 to about 34; from about 1 to about 24; and from about 1 to about14. In a second embodiment, the antibody of the invention binds one ormore of an epitope of the mature GDF8 protein (SEQ ID NO:340) withinamino acids from about 35 to about 109; from about 45 to about 109; fromabout 55 to about 109; from about 65 to about 109; from about 75 toabout 109; from about 85 to about 109; from about 92 to about 109; orfrom about 95 to about 109. In a third embodiment, the antibody orantigen-binding fragment of the antibody binds within an epitope of themature human GDF8 protein from about amino acid residue 48 to about 72;from about 48 to about 69; from about 48 to about 65; from about 52 toabout 72; from about 52 to about 65; or from about 56 to about 65.

According to certain embodiments of the invention, the anti-GDF8antibodies bind to human GDF8 but not to GDF8 from other species.Alternatively, the anti-GDF8 antibodies of the invention, in certainembodiments, bind to human GDF8 and to GDF8 from one or more non-humanspecies. For example, the anti-GDF8 antibodies of the invention may bindto human GDF8 and may bind or not bind, as the case may be, to one ormore of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit,goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesus orchimpanzee GDF8.

The invention encompasses a human or humanized anti-GDF8 monoclonalantibody conjugated to a therapeutic moiety (“immunoconjugate”), such asa cytotoxin, a chemotherapeutic drug, an immunosuppressant or aradioisotope. Cytotoxin agents include any agent that is detrimental tocells. Examples of suitable cytotoxin agents and chemotherapeutic agentsfor forming immunoconjugates are known in the art, see for example, WO05/103081, which is herein specifically incorporated by reference).

The antibodies of the invention may be monospecific, bi-specific, ormultispecific. Multispecific antibodies may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for more than one target polypeptide. See, e.g., Tuttet al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, TrendsBiotechnol. 22:238-244. The anti-GDF8 antibodies of the invention can belinked to or co-expressed with another functional molecule, e.g.,another peptide or protein. For example, an antibody or fragment thereofcan be functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment to produce abi-specific or a multispecific antibody with a second bindingspecificity. For example, the invention includes bi-specific antibodieswherein one arm of an immunoglobulin is specific for human GDF8 or afragment thereof, and the other arm of the immunoglobulin is specificfor a second therapeutic target or is conjugated to a therapeuticmoiety.

An exemplary bi-specific antibody format that can be used in the contextof the invention involves the use of a first immunoglobulin (Ig) CH3domain and a second Ig CH3 domain, wherein the first and second Ig CH3domains differ from one another by at least one amino acid, and whereinat least one amino acid difference reduces binding of the bispecificantibody to Protein A as compared to a bi-specific antibody lacking theamino acid difference. In one embodiment, the first Ig CH3 domain bindsProtein A and the second Ig CH3 domain contains a mutation that reducesor abolishes Protein A binding such as an H95R modification (by IMGTexon numbering; H435R by EU numbering). The second CH3 may furthercomprise a Y96F modification (by IMGT; Y436F by EU). Furthermodifications that may be found within the second CH3 include: D16E,L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N,V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, andV82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT;Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the caseof IgG4 antibodies. Variations on the bi-specific antibody formatdescribed above are contemplated within the scope of the invention.

The anti-GDF8 antibodies and antibody fragments of the inventionencompass proteins having amino acid sequences that vary from those ofthe described antibodies, but that retain the ability to bind humanGDF8. Such variant antibodies and antibody fragments comprise one ormore additions, deletions, or substitutions of amino acids when comparedto parent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, the anti-GDF8antibody-encoding DNA sequences of the invention encompass sequencesthat comprise one or more additions, deletions, or substitutions ofnucleotides when compared to the disclosed sequence, but that encode ananti-GDF8 antibody or antibody fragment that is essentiallybioequivalent to an anti-GDF8 antibody or antibody fragment of theinvention. Examples of such variant amino acid and DNA sequences arediscussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-GDF8 antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-GDF8 antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising theantibodies or antigen-binding fragments thereof of the invention. Theadministration of therapeutic compositions in accordance with theinvention will be administered with suitable carriers, excipients, andother agents that are incorporated into formulations to provide improvedtransfer, delivery, tolerance, and the like.

A multitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-waterand water-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. Any of the foregoing mixtures may be appropriate intreatments and therapies in accordance with the invention, provided thatthe active ingredient in the formulation is not inactivated by theformulation and the formulation is physiologically compatible andtolerable with the route of administration. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to beadministered, target disease, conditions, route of administration, andthe like. When the antibody of the invention is used for treatingvarious conditions and diseases associated with GDF8, in an adult, it isadvantageous to administer the antibody of the invention normally at asingle dose of about 0.01 to about 20 mg/kg body weight, about 0.1 toabout 10 mg/kg body weight, or about 0.1 to about 5 mg/kg body weight.Alternatively, the antibody or antigen binding fragment thereof can beadministered to a healthy subject in combination with an exerciseregimen. In embodiments, the healthy subject is experiencing age relatedloss of lean muscle mass and/or post-surgical muscle wasting. Inembodiments, an effective amount for a subject is at least 400 mg, orabout 36 mg/kg (assuming an average of 70 kg for an adult human).

Depending on the severity of the condition, the frequency and theduration of the treatment can be adjusted. In other parenteraladministration and oral administration, the antibody can be administeredin a dose corresponding to the dose given above. When the condition isespecially severe, the dose may be increased according to the conditionup to the amount that causes significant side effects, if any. Inembodiments, the subject receives at least two doses or moresubcutaneously. In embodiments, the subjects receive multiple dosesintermittently, for example, once every week, once every two weeks, onceevery three weeks, once every four weeks, once every five weeks, andonce every six weeks

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533).

A pharmaceutical composition of the invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe invention. Such a pen delivery device can be reusable or disposable.A reusable pen delivery device generally utilizes a replaceablecartridge that contains a pharmaceutical composition. Once all of thepharmaceutical composition within the cartridge has been administeredand the cartridge is empty, the empty cartridge can readily be discardedand replaced with a new cartridge that contains the pharmaceuticalcomposition. The pen delivery device can then be reused. In a disposablepen delivery device, there is no replaceable cartridge. Rather, thedisposable pen delivery device comes prefilled with the pharmaceuticalcomposition held in a reservoir within the device. Once the reservoir isemptied of the pharmaceutical composition, the entire device isdiscarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the invention. Examples include, but are not limited toAUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system, for example, with the use of a pump orpolymeric materials. In another embodiment, a controlled release systemcan be placed in proximity of the composition's target, thus requiringonly a fraction of the systemic dose.

Examples of the composition for oral administration include solid orliquid dosage forms, specifically, tablets (including dragees andfilm-coated tablets), pills, granules, powdery preparations, capsules(including soft capsules), syrup, emulsions, suspensions, etc. Such acomposition is manufactured by publicly known methods and contains avehicle, a diluent or an excipient conventionally used in the field ofpharmaceutical preparations. Examples of the vehicle or excipient fortablets are lactose, starch, sucrose, magnesium stearate, and the like.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to 500 mg per dosage form in a unit dose;especially in the form of injection, it is preferred that the aforesaidantibody is contained in about 5 to 500 mg and in about 10 to 400 mg forthe other dosage forms.

Therapeutic Uses of the Antibodies

The antibodies of the invention are useful, inter alia, for increasinglean muscle mass in a subject. In some embodiments, a method ofincreasing lean muscle mass comprises providing an exercise regimen forthe subject, and administering a composition comprising an effectiveamount of a GDF-8 inhibitor. In embodiments, the subject is a subjectthat has not been placed on exercise limitation by a physician and/ordoes not have a condition or disorder for which exercise iscontraindicated. In embodiments, a subject has a loss of lean musclemass. In other embodiments, the subject is at least 50 years, 55 years,or 60 years of age or older.

In embodiments, the antibodies of the invention are useful, inter alia,for the treatment, prevention and/or amelioration of any disease ordisorder associated with GDF8 activity. More specifically, theantibodies of the invention are useful for the treatment of anycondition or affliction which can be improved by increasing musclestrength/power and/or muscle mass and/or muscle function in anindividual, or by favorably altering metabolism (carbohydrate, lipid andprotein processing) by blocking GDF8 activity. Exemplary diseases,disorders and conditions that can be treated with the anti-GDF8antibodies of the invention include, but are not limited to, sarcopenia,cachexia (either idiopathic or secondary to other conditions, e.g.,cancer, chronic renal failure, or chronic obstructive pulmonarydisease), muscle injury, muscle wasting and muscle atrophy, e.g., muscleatrophy or wasting caused by or associated with disuse, immobilization,bed rest, injury, medical treatment or surgical intervention (e.g., hipfracture, hip replacement, knee replacement, etc.) or by necessity ofmechanical ventilation. Additional disorders that can be treated withthe anti-GDF8 antibodies of the invention include, but are not limitedto, sIBM (Sporadic Inclusion Body Myositis).

The invention includes therapeutic administration regimens whichcomprise administering an anti-GDF8 antibody of the invention incombination with at least one additional therapeutically activecomponent. Non-limiting examples of such additional therapeuticallyactive components include other GDF8 antagonists (e.g., small moleculeinhibitors of GDF8 or other GDF8 antibodies or binding molecules),growth factor inhibitors, immunosuppressants, anti-inflammatory agents,metabolic inhibitors, enzyme inhibitors, and cytotoxic/cytostaticagents. The additional therapeutically active component(s) may beadministered prior to, concurrent with, or after the administration ofthe anti-GDF8 antibody of the invention.

In a related embodiment, a method comprises combining administration ofan antibody or antigen binding fragment thereof to a subject that isexercising on a regular basis, such as weight bearing exercises. Inembodiments, a method comprises providing an exercise regimen, such as aregimen that promotes an increase in lean muscle mass. In embodiments,the exercise regimen includes one or more of resistance training,strength training, pilates, aerobic exercise, weight training, and yoga.In a specific embodiment, the exercise regimen includes resistancetraining and/or weight training. In embodiments, an exercise regimen isprovided in writing and/or by illustration, on a computer readablemedium, by video, or in an exercise facility.

In embodiments, resistance training includes training of any muscles ofthe body including without limitation, the muscles of a non-dominanthand, a dominant hand, a leg, an arm, a back, an abdominal muscle, aquadricep, a calf, a bicep, a tricep, a shoulder, a gluteus muscle,and/or a chest muscle. Resistance training can include the use ofweights, a resistance band, exercise machines, and/or the subject's ownbody weight. Exercises can include chest press, leg press, arm curl, legcurl, hand grip, abdominal crunch, calf press, bicep curl, tricep curl,plank, side plank, and/or stair climb.

In embodiment, the exercise regimen provides for at least one set ofexercises per muscle group and in specific embodiments, more than one,two or three sets of exercises. In embodiments, a set of exercisesinclude at least eight repetitions or more.

A subject follows the provided exercise regimen at least once a week,twice a week, three times a week, four times a week, or five times aweek. In embodiments, the exercise regimen is followed for at least 12weeks.

In embodiments, a subject follows a set of exercises at a minimal levelof intensity, for example, at 50% or less of the maximum weight that canbe lifted for that exercise (1RM). In embodiments, the intensity isprogressively increased during the exercise regimen. In embodiments, a1-RM for each exercise (chest press, leg press, leg curl, and arm curl)can be measured at the exercise facility on the equipment used forexercise training at various time intervals such as baseline, week 4,and week 8. In embodiments, intensity is increased about 10% of the 1RMper week. In embodiments, intensity is at least 50%, and is maintainedat about 65-90% of maximum.

In embodiments, a method further comprises monitoring the subjectsresistance training comprising providing a device that measures trainingand/or providing a log book or computer program for tracking therepetitions, the duration, the intensity, and the frequency of theresistance training. In embodiments, a device includes an accelerometer,a dynamometer, a linear positioning device, and an actigraph.

In embodiments, a method comprises administering a GDF-8 inhibitor in anamount effective to increase lean muscle mass without resulting inadverse side effects on cardiac muscle and/or function. As discussedpreviously, GDF-8 inhibitors could be associated with cardiachypertrophy. However, in embodiments, a dose and administration regimenis selected to minimize any effects on cardiac muscle and/or function.In embodiments, markers such as creatinine kinase, troponin, and thelike are monitored during treatment. In embodiments, the treatment isdiscontinued if cardiac hypertrophy is observed, and/or if cardiacmarkers indicative of cardiac damage are elevated at least 20%.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1. Generation of Human Antibodies to Human GDF-8

Human anti-GDF8 antibodies were generated as described in U.S. Pat. No.8,840,894. The exemplary GDF8 inhibitor used in the following Example isthe human anti-GDF-8 antibody designated “H4H1657N2” (also referred toas REGN1033). H4H1657N2 has the following amino acid sequencecharacteristics: heavy chain variable region (HCVR) comprising SEQ IDNO:360; light chain variable domain (LCVR) comprising SEQ ID NO:368. TheCDRs of REGN1033 comprise heavy chain complementarity determining region1 (HCDR1) comprising SEQ ID NO:362; HCDR2 comprising SEQ ID NO:364;HCDR3 comprising SEQ ID NO:366; light chain complementarity determiningregion 1 (LCDR1) comprising SEQ ID NO:370; LCDR2 comprising SEQ IDNO:372; and LCDR3 comprising SEQ ID NO:374.

Example 2: Clinical Trial of Safety and Bioeffect of Anti-GDF-8 with andwithout Exercise

A randomized, double-blind, placebo-controlled, multicenter,parallel-group study of repeated doses of subcutaneous REGN1033treatment effects on safety, body composition and muscle volume, musclestrength and stair climb function in 120 healthy male and femalesubjects with a sedentary lifestyle, who were 60 years of age and olderwas conducted. A 2×2 factorial design was used; up to 120 subjects (4arms of 30 subjects each) were to be randomized in a 1:1:1:1 ratio toPlacebo alone, REGN1033 (400 mg SC Q2W×6 doses) alone,Placebo+resistance exercise training (RT), or REGN1033+RT. Randomizationwas stratified by sex and by study site. The RT consisted of acenter-based low intensity resistance exercise training at 50% of1-repetition maximum (1-RM) twice a week for a total of 12 weeks with atleast one progressive adjustment of exercise load during the study.

The primary objective of the study was to assess the effect of REGN1033,with and without exercise, on total lean body mass as measured bydual-energy x-ray absorptiometry (DEXA). Secondary objects includeassessments of effects on safety and tolerability; effects onappendicular lean mass and fat mass by DEXA, effects on thigh musclevolume measured by MRI, upper and lower body strength by 1-repetitionmaximum methods, maximum hand grip strength, and stair climb power.

Patient Selection

The target population for this study 120 healthy male and femalesubjects with a sedentary lifestyle, who were 60 years of age and older.

The inclusion criteria were as follows: 1. Men and women aged 60 yearsand older, with no significant health issues or conditions; 2. Sexuallyactive males willing to use contraceptives and not to donate spermduring the study and through 4 months after the study; 3. Femalesrequire clinical confirmation of postmenopausal status (at least 12months since last menses, confirmed by postmenopausal levels of FSH>20mIU/ml, or surgically sterile); 4. Body mass index (BMI) between 19 and35 kg/m2 inclusive; 5. With no condition that could limit participationin supervised resistance training exercise based on the PAR-Q; 6.Sedentary lifestyle defined by a score of <125 on the CHAMPS-18 physicalactivity questionnaire in the last 3 months; 7. Willing to maintaincurrent diet and adhere to exercise programs described for the study andto not start any new dieting/weight management programs; 8. Willing andable to return for all clinic visits and complete all study-relatedprocedures; 9. Provide complete study-related questionnaires; and 10.Provide the informed consent form (ICF).

The exclusion criteria for the study include those subjects that havebeen hospitalized or had major surgery, have osteoarthritis,rheumatologic diseases or orthopedic disorders which limit joint rangeof motion or ability to exercise; have gastrointestinal disorders,chronic kidney disease, cancer, pulmonary disease, cardiac disease,asthma, stroke with residual paresis, paralysis, multiple sclerosis,Parkinson Disease, cognitive impairment, psychiatric conditions thatwarrant acute or chronic therapeutic intervention (eg, major depressivedisorder, bipolar disorder, panic disorder, schizophrenia), current orprevious use of any drugs known to influence muscle mass or performancewithin 6 months, and unable to undergo MRI of the thighs

Drug Administration

REGN1033 was supplied as a lyophilized drug product and was administeredsubcutaneously in this study. Each vial of lyophilized REGN1033 wasreconstituted under aseptic conditions to a final concentration of 100mg/mL. Placebo matching REGN1033 is prepared in the same formulation asREGN1033 without the addition of REGN1033. The volume for placebo wasthe same at each dose level. Subjects received 400 mg REGN1033 orplacebo (combined with resistance training RT for subjects in groups 3and 4) every 2 weeks for a total of 6 doses SC in the abdomen. Thespecific abdominal quadrant was documented.

Study Design

This study had a screening period of 28 days (day −28 to day −1,screening/pretreatment), a drug treatment period (day 1 to day 71) and a10 week follow up period after the last dose administration. Baselinemeasures for weight, strength measures and stair climb function, andechocardiography, were obtained during the screening period (day −14 today −1). Baseline measures for DEXA and MRI were obtained between day−7±3 and day −1. For all other parameters, baseline measures wereobtained on day 1 (baseline). The total duration of the study from firstdose administration was approximately 20 weeks.

Subjects were screened from day −28 to day −1 and eligible subjects wererandomized to 1 of the 4 groups on day 1: Placebo; Placebo and RT;REGN1033; REGN1033 and RT. Subject eligibility was determined bystandard screening procedures as well as screening for cognitiveimpairment using the Mini-Mental State Examination (MMSE) questionnaire,and screening for a sedentary life-style using the Community HealthActivities Model Program for Seniors (CHAMPS) 18 physical activityquestionnaire.

Within 2 weeks prior to the start of dosing (day −14 to day −1) allsubjects were familiarized with the exercises and muscle strength andfunction measurements: leg press, chest press, leg curl, arm curl,handgrip strength, and unloaded and loaded stair climb (8-steps).Baseline upper and lower body strength (as determined by 1-repetitionmaximum [1-RM] for chest press and leg press), maximum handgripstrength, and unloaded and loaded stair climb power were determined forall subjects within 2 weeks (day −14 to day −1) prior to the start ofdosing. For each strength/function measure, 2 tests were conducted forsubjects in all 4 treatment groups in 2 separate visits within 2 weeksbefore administration of the first dose of study drug to accommodatefamiliarization with and learning of the testing procedures. The averagevalue of these 2 measures during these 2 testing sessions will be usedas the baseline value.

The 1-RM for each RT exercise (chest press, leg press, leg curl, and armcurl) was measured at the exercise facility on the equipment was usedfor exercise training at baseline, week 4, and week 8 and which was usedto calculate the load for RT training during weeks 1 to 4, 5 to 8, and 9to 12. Exercise training was conducted twice weekly for the 12 weektreatment period. At least 1 day separated each exercise session. Within10 days prior to the start of dosing (day −7±3 days) all subjectsunderwent whole body DEXA to determine total and regional fat mass andappendicular lean mass. Subjects also underwent MRI of both thighs todetermine muscle volume and SC and intramuscular fat. The valuesobtained served as the baseline for these parameters.

On day 1, and every 2 weeks thereafter for a total of 6 times, subjectsreceived SC doses of study drug (400 mg of REGN1033 or placebo)administered in the abdomen as 2 injections of 2 ml each injection site.Subjects were observed for 30 minutes for vital signs and collection ofadverse events (AEs), including occurrence of injection site reactions.Accelerometry was used to monitor subject's physical activity during thestudy.

Subjects in groups 3 and 4 returned to the site for supervisedlow-intensity resistance training of the major muscle groups of theupper and lower extremities using resistance training equipment twice aweek for 12 weeks. All training exercises were performed at a relativelylow intensity of 50% of the 1-RM for each exercise.

All subjects completing the study returned for laboratory and safetyassessments at weeks 2, 4, 6, 8 and 10 on days 15, 29, 43, 57, and 71(all with a visit window of 3 days) when subsequent doses of study drugwere administered. Subjects were followed for 10 weeks after the lastdose administration (day 71) and returned to the clinic for laboratoryand safety assessments at weeks 12, 14, 16, and 20 (days 85±3, 99±3,113±3, and 141±3 [until the end of the last study visit]). Efficacymeasures (DEXA, MRI, muscle strength and physical function) wereobtained at screening and at week 12 (day 85±3). DEXA and musclestrength (double leg press, chest press, and maximum handgrip) andphysical function (stair climb power) were determined at week 6 and week20. Electrocardiograms were administered at day 1, week 4, week 8, week12 and at the end of the study. Echocardiograms were conducted at week12 (day 853 days).

All subjects were instructed to wear a GT3X Actigraph device (set at 30Hz sampling rate) on the hip for the first 2 weeks on study from day 1until the day 15 visit and another 14 day period between day 71 and day85 (excluding the periods for center-resistance training exercise andduring sleep). Subjects returned the device to the study site fordownloading of data (day 15 and day 85). Time spent at various activitylevels was determined based on activity threshold analysis usingpre-defined cut-offs. Estimates of metabolic rates and energyexpenditure were determined by predefined algorithms (Actigraph Corp).

Sample Analysis and Statistics

The primary efficacy variable, i.e. the percent change in Total LeanMass by dual energy x-ray absorptiometry (DEXA) from baseline to week12, was analyzed using a mixed-effect model repeated measure (MMRM)approach. Secondary efficacy variables were analyzed in a similarfashion. The model included factors (fixed effects) for treatment (with4 levels of R1033+RT, R1033 without RT, Placebo+RT and Placebo withoutRT), baseline stratum (gender), visit, baseline value andtreatment-by-visit interaction as covariates. The comparison of theprimary endpoint between REGN1033 without RT and Placebo without RT wasmade. Same comparison was made between REGN1033 with RT and Placebo withRT. The exercise effect was also explored.

Results

Across all 4 treatment groups, 93.8-96.6% of randomized subjectscompleted their Week 12 visit. There was only one subject, in thePlacebo alone treatment group, who discontinued the study due to anadverse event. The % of subjects in each of Placebo (PLC), Placebo+RT(PLC+RT), REGN1033(R1033), and REGN1033+RT (R1033+RT) groups thatreceived all 6 doses of study drug was: 87.5%, 93.1%, 84.4%, and 90.6%,respectively. There was no imbalance across groups in compliance witheither treatment or RT. The demographics were balanced among the 4groups except the Placebo group had a higher percentage (18.8%) of blacksubjects; and the Placebo+RT group had higher percentage (75.9%) and theREGN1033 group had lower percentage (56.3%) of subjects' age greaterthan 65 years. The baseline values for DEXA, MRI, muscle strength andphysical function for each group did not significantly differ from oneanother. (data not shown)

The primary objective of the study was to assess the effect of REGN1033,with and without exercise, on total lean body mass as measured bydual-energy x-ray absorptiometry (DEXA). Secondary objects includeassessments of effects on safety and tolerability; effects onappendicular lean mass and fat mass by DEXA, effects on thigh musclevolume measured by MRI, upper and lower body strength by 1-repetitionmaximum methods, maximum hand grip strength, and stair climb power. Inaddition, potential metabolic effects of REGN1033 on HbA1C and HOMA-IRwere examined.

The primary efficacy variable, i.e. the percent change in Total LeanMass by dual energy x-ray absorptiometry (DEXA) from baseline to week12, was analyzed using a mixed-effect model repeated measure (MMRM)approach. The model included factors (fixed effects) for treatment (with4 levels of R1033+RT, R1033 without RT, Placebo+RT and Placebo withoutRT), baseline stratum (gender), visit, baseline value andtreatment-by-visit interaction as covariates. The comparison of theprimary endpoint between REGN1033 without RT and Placebo without RT wasmade. Same comparison was made between REGN1033 with RT and Placebo withRT. The exercise effect was also explored. Secondary Efficacy Endpointswith continuous outcome were analyzed by a similar method for primaryefficacy endpoint.

The doses and dose regimens for this study were selected based on theresults from other studies.

In a completed SAD study, 76 healthy volunteers received REGN1033 atdoses up to 10 mg/kg intravenously (IV) and 400 mg SC. Among these, anelderly cohort of 8 healthy volunteers 65 to 85 years old received 6mg/kg IV of study drug. All doses were well tolerated; no clinicallysignificant safety signals were observed. (data not shown)

Another study was designed to assesses the safety, tolerability,pharmacokinetics (PK), immunogenicity, and pharmacodynamic (PD) effectsof REGN1033 administered SC in healthy volunteers 60 years of age andolder. A total of 5 cohorts with 12 subjects enrolled in each cohortwere studied. Subjects received SC doses of REGN1033 (n=9) or placebo(n=3). The planned REGN1033 dose regimens were 100, 200, or 400 mg Q2Wfor a total of 6 doses per subject and 200 mg or 400 mg Q4W for a totalof 3 doses per subject. The results from the study, shown in FIG. 1A,suggest that these doses of REGN1033 are well tolerated and areassociated with an increase in lean mass detected by DEXA.

From these studies, PK and total GDF8 PD data suggest that REGN1033 mayachieve concentrations associated with saturation of target engagementthroughout the dose interval for the 400 mg Q4W and 400 mg Q2W doselevels. At the 100 mg Q4W dose level, there is expected to be REGN1033exposures clearly less than those achieved at the higher doses. PK andPD data from phase 1 studies suggest that this lower dose will notachieve concentrations associated with engagement of the target acrossthe entire dose interval in all individuals.

The results of the outcome of the primary efficacy variable are shown inTable 1 and FIG. 1B.

TABLE 1 Week 12 MMRM Week 12 Week 12 REGN Week 12 Week 12 MMRM MMRMmodel REGN REGN + combined PBO + REGN + model model REGN^(‡) alone vs.RT vs. vs. PBO RT vs. RT vs. REGN Exercise Exercise PBO alone PBO + RTcombined PBO REGN effect Effect Interaction Summary of Primary EfficacyPercent change in total lean mass 1.66% vs. 3.10% vs. 2.39% vs. −0.25%vs. 3.10% vs. (p = (p = (p = by dual energy x-ray 1.79% −0.25% 0.79%1.79% 1.66% 0.0008) 0.6838) 0.0124) absorptiometry (DXA) from (p = (p <(p = (p = (p = baseline 0.8640) 0.0001) 0.0048) 0.0088) 0.0609)

Without exercise, REGN1033 (REGN) treatment did not increase total leanbody mass measured by DEXA compared with Placebo (PBO) treatment(placebo adjusted change −0.13% p=0.8640, from baseline to week 12).With exercise, REGN1033 (REGN+RT) treatment significantly increasedtotal lean body mass measured by DEXA from baseline to week 12 comparedwith Placebo (PBO+RT) treatment (placebo adjusted increase of 3.34%,p<0.0001). See Table 1 and FIG. 1B. The percent change in the‘Placebo+RT’ group is significantly lower than in the ‘Placebo alone’group: −0.25% vs. 1.79% (p=0.0088), but the ‘REGN1033+RT’ group isnumerically higher than the ‘REGN1033 alone’ group: 3.10% vs. 1.66%(p=0.0609).

The factorial MMRM model also shows a significant REGN1033 treatmenteffect on percentage change of total lean body mass by DEXA frombaseline to week 12 (P=0.0008) which is sustained at week 20 (P=0.0004).In addition, MMRM also shows an interaction effect (p=0.0124) betweenExercise and Treatment.

In subjects randomized to receive progressive resistance trainingexercise, REGN1033(REGN+RT) treatment significantly increased theappendicular lean body mass measured by DEXA from baseline to week 12compared with Placebo (PBO+RT) treatment (LS Mean difference vs Placebo4.34%, P=0.001). As observed in the analysis of total lean body mass,the percent change in appendicular lean mass in the Placebo+RT group wassignificantly lower than in the Placebo alone group: 0.08% vs. 2.25%(p=0.0200), but the REGN1033+RT group was significantly higher than theREGN1033 alone group: 4.42% vs. 2.30% (p=0.0217). The factorial MMRMmodel also shows a significant treatment effect (0.0008) and interactionbetween treatment and exercise at week 12 (p=0.0040) which are sustainedat week 20. See Table 2 and FIG. 2.

TABLE 2 Week 12 MMRM Week 12 Week 12 REGN Week 12 Week 12 MMRM MMRMmodel REGN REGN + combined PBO + REGN + model model REGN^(‡) alone vs.RT vs. vs. PBO RT vs. RT vs. REGN Exercise Exercise PBO alone PBO + RTcombined PBO REGN effect Effect Interaction Percent change in 2.30% vs.4.42% vs. 3.37% vs. 0.08% vs. 4.42% vs. (p = (p = (p = Appendicular leanmass (g) by 2.25% 0.08% 1.19% 2.25% 2.30% 0.0008) 0.9728) 0.0040) DXA d= d = d = (p = (p = 0.04% 4.34% 2.18% 0.0200) 0.0217) (p = (p < (p =0.9617) 0.0001) 0.0015).

REGN1033 treatment, with or without exercise, resulted in statisticallysignificant increases in MRI thigh muscle volume. See FIGS. 3A and 3B.The placebo adjusted effects on MRI thigh muscle volume (excludingintramuscular fat and vessels) at week 12 was +3.7%, p=0.0006 withoutexercise (REGN alone) and +4.5%, p<0.0001 with exercise (REGN+RT). Thereis a trend of increase of thigh muscle including intramuscular fat andvessels volume in the REGN1033 alone group compared to Placebo alonegroup and an increase of thigh muscle including intramuscular fat andvessels volume in the REGN1033+RT group as compared with Placebo+RTgroup. At week 12, the LS Mean percentage (%) change from baseline ofthigh muscle including intramuscular fat and vessels volume (originalscale) in Placebo alone, Placebo+RT, REGN1033 alone, and REGN1033+RTgroups are −0.03%, −0.434%, 2.076%, and 3.405% respectively. In subjectsrandomized to not receive exercise, REGN1033 treatment significantlyincreased the thigh muscle including intramuscular fat and vesselsvolume measured by MRI from baseline to week 12 compared with Placebotreatment (LS Mean difference vs Placebo 2.11%%, P=0.0139). In subjectsrandomized to receive progressive resistance training exercise, REGN1033treatment significantly increased the thigh muscle includingintramuscular fat and vessels volume measured by MRI from baseline toweek 12 compared with Placebo treatment (LS Mean difference vs Placebo3.84%, P<0.0001).

The factorial MMRM model also shows a significant treatment effect(<0.0001) but not exercise effect (p=0.4453). The interaction betweentreatment and exercise at week 12 is not significant (p=0.1651). Therewas an increase of lean body mass by DEXA measures in the placebo groupwhich was not accompanied by an increase of muscle volume by MRImeasures. The reason for this discrepancy is unknown.

There is a trend of increase of Gynoid Fat Mass in the Placebo alonegroup at week 12 compared to other treatment groups and a decrease ofGynoid Fat Mass in the REGN1033+RT group. At week 12, the LS Meanpercentage (%) change from baseline in Placebo alone, Placebo+RT,REGN1033 alone, and REGN1033+RT groups are 2.42%, −0.49%, −0.27%, and−2.17% respectively. See FIG. 4. The factorial MMRM model shows trendsignificant treatment (p=0.0824) and exercise (0.0531) effects on gynoidfat mass but no interaction between Exercise and Treatment (p=0.8774).

Data from four muscle strength measures were also obtained. Equipmentwas standardized across all study sites for the chest press and legpress 1-RM measures and not for arm curl and leg curl measures.Furthermore, only a subset of subjects had arm curl and leg curlmeasures.

There was a trend of increase of leg press strength in all groups. Atweek 12, the LS Mean percentage (%) change from baseline in Placeboalone, Placebo+RT, REGN1033 alone, and REGN1033+RT groups are 4.7%,8.9%, 6.7%, and 9.7% respectively. The factorial MMRM model did not showsignificant treatment effect (p=0.8389) but a borderline exercise effect(p=0.0541) on leg press. The interaction effect between Exercise andTreatment is not significant (p=0.8029).

There was a trend of increase of chest press strength in all groups. TheREGN1033 alone group increased more as compared to Placebo alone group,and the REGN1033+RT group also increased more as compared to Placebo+RT.See FIG. 5. At week 12, the LS Mean percentage (%) change from baselinein Placebo alone, Placebo+RT, REGN1033 alone, and REGN1033+RT groups are2.2%, 10.5%, 10.4%, and 15.3% respectively. In subjects randomized tonot receive progressive resistance training exercise, REGN1033 treatmentincreased chess press strength from baseline to week 12 compared withPlacebo treatment (LS Mean difference vs Placebo 8.2%, P=0.0524). Insubjects randomized to receive progressive resistance training exercise,REGN1033 treatment numerically increased chest press from baseline toweek 12 compared with Placebo treatment, but not significant (LS Meandifference vs Placebo 5.3%, P=0.2316). The factorial MMRM model showed asignificant treatment effects (p=0.0525), and a statisticallysignificant exercise effect (0.0272) on chest press and no interactioneffect (p=0.5089) between Exercise and Treatment.

There was a trend of increase of leg curl strength in all groups up toweek 12. At week 12, the LS Mean percentage (%) change from baseline inPlacebo alone, Placebo+RT, REGN1033 alone, and REGN1033+RT groups were5.3%, 16.3%, 10.4%, and 9.7% respectively. The change in leg curlstrength REGN1033 alone was 5.1% higher than Placebo alone, however,REGN1033+RT was 6.6% lower than Placebo+RT. The percent change in thePlacebo+RT group was significant higher than in the ‘Placebo alone’group: 16.3% vs. 5.30% (p=0.0050), however the REGN1033+RT group was nothigher than the REGN1033 alone group: 9.79% vs. 10.4% (p=0.8388). Thefactorial MMRM model did not show a significant interaction effect(p=0.0229) between Exercise and Treatment.

There was a trend of increase of arm curl in all groups. (data notshown). At week 12, the LS Mean percentage (%) change from baseline inPlacebo alone, Placebo+RT, REGN1033 alone, and REGN1033+RT groups are8.1%, 21.7%, 12.4%, and 15.7% respectively. In subjects randomized tonot receive progressive resistance training exercise, REGN1033 treatmentincreased arm curl strength from baseline to week 12 compared withPlacebo, but this difference was not statistically significant (LS Meandifference vs Placebo 4.3%, P=0.4589).

In subjects randomized to receive progressive resistance trainingexercise, REGN1033 treatment increased less in arm curl strength frombaseline to week 12 compared with Placebo, but not significant (LS Meandifference vs Placebo −6.0%, P=0.3121). The factorial MMRM model showedno significant REGN1033 treatment effect (p=0.8958), a borderlineexercise (p=0.053) effects, and no significant interaction effect(p=0.1318) between Exercise and Treatment.

There was a trend of increase of the dominate hand grip strength inREGN1033 groups. (data not shown). At week 12, the LS Mean percentage(%) change from baseline in Placebo alone, Placebo+RT, REGN1033 alone,and REGN1033+RT groups were 1.8%, 1.2%, 4.8%, and 5.0% respectively. Insubjects randomized to not receive progressive resistance trainingexercise, REGN1033 treatment increased the dominate hand grip strengthfrom baseline to week 12 compared with Placebo, but not significant (LSMean difference vs Placebo 3.0%, P=0.4180). In subjects randomized toreceive progressive resistance training exercise, REGN1033 treatmentincreased more in the dominate hand grip strength from baseline to week12 compared with Placebo, but not significant (LS Mean difference vsPlacebo 3.8%, P=0.3198). The factorial MMRM model shows a statisticallysignificant REGN1033 treatment effect (p=0.0407), but no exercise effect(p=0.7033) or interaction between Exercise and Treatment (p=0.8283).

There was a trend of increase of the non-dominate hand grip strength inREGN1033 groups but not in Placebo groups. (data not shown). At week 12,the LS Mean percentage (%) change from baseline in Placebo alone,Placebo+RT, REGN1033 alone, and REGN1033+RT groups were 1.8%, −0.8%,7.0%, and 4.2% respectively. In subjects randomized to not receiveprogressive resistance training exercise, REGN1033 treatment increasedmore in the non-dominate hand grip strength from baseline to week 12compared with Placebo, but not significant (LS Mean difference vsPlacebo 5.1%, P=0.1432). In subjects randomized to receive progressiveresistance training exercise, REGN1033 treatment increased more in thenon-dominate hand grip strength from baseline to week 12 compared withPlacebo, but not significant (LS Mean difference vs Placebo 5.0%,P=0.1708). The factorial MMRM model showed a statistically significantREGN1033 treatment effect (p=0.0039) and a borderline significanceexercise effect (p=0.0581) on the non-dominate hand grip strength. Therewas no interaction effect between Exercise and Treatment (p=0.6224).

There was a trend of increase of loaded stair climb power in all groups.(data not shown) At week 12, the LS Mean percentage (%) change frombaseline in Placebo alone, Placebo+RT, REGN1033 alone, and REGN1033+RTgroups were 5.6%, 12.9%, 12.2%, and 15.0% respectively. In subjectsrandomized to not receive progressive resistance training exercise,REGN1033 treatment increased more in the loaded stair climb power frombaseline to week 12 compared with Placebo, but not significant (LS Meandifference vs Placebo 6.6%, P=0.1303). In subjects randomized to receiveprogressive resistance training exercise, REGN1033 treatment increasedmore in loaded stair climb power from baseline to week 12 compared withPlacebo, but not significant (LS Mean difference vs Placebo 2.2%,P=0.6368). The factorial MMRM model did not show a statisticallysignificant treatment effect (p=0.3612) or interaction between Exerciseand Treatment (p=0.8043), but showed a statistically significantexercise effect (p=0.0151) in loaded stair climb.

There was a trend of increase of unloaded stair climb power in allgroups. At week 12, the LS Mean percentage (%) change from baseline inPlacebo alone, Placebo+RT, REGN1033 alone, and REGN1033+RT groups were6.1%, 12.5%, 5.4%, and 9.8% respectively. There was not differencebetween REGN1033 and the Placebo groups. The factorial MMRM model didnot show a statistically significant treatment effect (p=0.4670) orinteraction between Treatment and Exercise (p=0.4180) but showed astatistically significance Exercise effect (p=0.0295). The percentchange in the Placebo+RT group was higher than in the Placebo alonegroup 12.5% vs. 6.1% (p=0.0900), and the REGN1033+RT group is higherthan ‘REGN1033 alone’ group: 9.8% vs. 5.4% (p=0.2253), however thesedifferences are not statistically significant.

REGN1033 treatment and the resistance training (RT) exercises weregenerally well tolerated in the study. The results for Treatment AdverseEvents (TEAS) are shown in Table 3.

TABLE 3 REGN1033 Placebo 400 mg + Alone RT 400 mg RT (N = 32) (N = 29)(N = 32) (N = 32) Number of TEAEs 151  103  129  98  Number of seriousTEAEs 1 1 3 0 Subjects with at least one TEAE 26 (81.3%) 27 (93.1%) 27(84.4%) 28 (87.5%) Subjects with at least one drug related TEAE 10(31.3%)  6 (20.7%) 14 (43.8%) 10 (31.3%) Subjects with at least oneserious TEAE 1 (3.1%) 1 (3.4%) 2 (6.3%) 0 Subjects with TEAEs resultingin 3 (9.4%) 1 (3.4%) 1 (3.1%) 0 discontinuation of study drug Subjectswith a TEAE resulting in death 0 0 0 0

There were no deaths reported in this study and a total of 5 seriousTEAS occurred in 4 subjects, including foot fracture in 1 subject in thePlacebo Alone group; cholecystitis in 1 subject in the Placebo+RT group,stress cardiomyopathy in 1 subject in the REGN1033 group, andhypokalaemia and hypotension in 1 subject in the REGN1033 group. Noserious TEAS were reported in the REGN1033+RT group. Subjects in PlaceboAlone group reported more TEAEs and treatment-related TEAEs compared toother groups. The number and percentage of subjects experiencing atleast one TEAE were similar across all treatment groups: 26 (81.3%) inplacebo Alone, 27 (93.1%) in Placebo+RT, 27 (84.4%) in REGN1033, and 28(87.5%) in REGN1033+RT group, respectively. A total of 5 subjects (3 and1 in Placebo and Placebo+RT groups, compared to 1 and 0 subjects inREGN1033 and REGN1033+RT groups, respectively) discontinued drugtreatment due to TEAEs.

Across the potentially clinically significant values (PCSV) categoriesof vital signs, ECG and hematology, there was no finding of an imbalancesignaling higher frequency of PCSVs in the REGN1033 treatment groups.There were numerically more subjects with chemistry PCSVs in theREGN1033 Combined group than there were in the PLACEBO COMBINED group.Elevated Creatine Kinase values of >3×ULN occurred in 2 (6.3%). 1(3.4%), 2 (6.3%) and 4 (12.5%) of subjects in the Placebo alone,Placebo+RT, REGN1033, and REGN1033+RT groups, respectively. There wereno occurrence of Creatine Kinase values >10×ULN (Table 14.3.4.2.3). Moresubjects in the REGN1033 treated groups (7 [10.9%]) had a body weightincrease >5% than in the Placebo groups (3, [4.9%], Table 14.3.5.1.2).

A review of echocardiogram parameters related to cardiac structure andfunction did not reveal any signal of cardiac hypertrophy or any otherdeleterious effects on cardiac function. REGN1033 treatment had noeffect on left ventricular ejection fraction, LV wall thickness orinterventricular septum thickness. There were no reported increases inLV mass or LV mass index in either the REGN1033 or the Placebo treatmentgroups. (data not shown)

Conclusions

With exercise, REGN1033 treatment significantly increased total leanbody mass in healthy subjects measured by DEXA from baseline to week 12compared with Placebo treatment (3.1% vs. −0.2%, <0.0001). Withoutexercise, REGN1033 treatment did not significantly increase total leanbody mass measured by DEXA compared with Placebo treatment.

For secondary efficacy endpoints, the results of appendicular lean massby DEXA are consistent with those observed in total lean body mass byDEXA. REGN1033 treatment, with or without exercise, resulted instatistically significant increases in MRI thigh muscle volume excludingintramuscular fat and vessels week 12 compared with Placebo treatment(placebo adjusted increase of 3.7%, P=0.0006 without exercise and 4.5%,P<0.0001 with exercise).

The resistance training (RT) exercises in this study were well toleratedin this elderly population. Significant exercise effects were shown byMMRM model in majority of the strength/function measures. This level ofRT exercise did not result in significant increase of lean mass measuredby DEXA or muscle volume measured by MRI. REGN1033 treatment alsotrended toward positive effects on several strength and functionalendpoints examined in this study. Positive effects were seen in-chestpress strength, handgrip strength, and loaded stair climb function.

Overall, REGN1033 SC 400 mg Q2W was generally well tolerated in thistrial. The numbers of patients reporting TEAEs were comparable acrosstreatment groups. A review of TEAEs did not reveal significant safetysignal. Echocardiogram examinations did not reveal deleterious effectson cardiac structure or function.

Example 3: Clinical Trial Protocol of Anti-GDF-8 Treatment of Subjectswith Sarcopenia with and without Exercise

A randomized, double-blind, placebo-controlled, multicenter phase 2study of the safety and efficacy of 3-month SC REGN1033 treatment inpatients with sarcopenia was conducted. Two hundred fifty patients wereenrolled, in 4 treatment groups. Eligible patients were males andfemales with sarcopenia and associated mobility impairment, 70 years ofage and older, with an average age of 78 years old. Patients wererandomized in a 1:1:1:1 ratio to receive placebo SC every 2 weeks (Q2W)for a total of 6 treatments, REGN1033 at 300 mg SC Q2W for a total of 6treatments, REGN1033 at 300 mg SC every 4 weeks (Q4W) for a total of 3treatments (with placebo on alternating weeks), and REGN1033 at 100 mgSC Q4W for a total of 3 treatments (with placebo on alternating weeks).The study had a screening/pretreatment period (day −28 to day −1), a12-week treatment period (day 1 to day 85), and an 8-week follow-upperiod (through day 141).

Screening and Pretreatment Procedures (Day −28 to Day −1)

A sequential screening process took place across 3 visits, with initialeligibility determined at visit 1, and pretreatment procedures performedat visit 2 and visit 3. If feasible at the sites, visit 1 and visit 2were conducted at the same time—if so, the visit 2 procedures wereperformed within 21 days of the first dose of study drug. Initialeligibility was determined at visit 1 by standard screening procedures,as well as 4-meter [4M] gait speed and the Mini-Mental State Examination(MMSE) score. Patients who met the initial eligibility criteria returnedto the clinic at visit 2 and visit 3 for pretreatment baselineprocedures and measurements.

The procedures included standard safety and laboratory assessments, DEXAscans, echocardiograms, strength measures (leg press, chest press, andhandgrip strength), and function measures (stair climb, Short PhysicalPerformance Battery [SPPB], 4M gait speed, and 6-Minute WalkTest[6MWT]).

Treatment Period and Study Drug Administration (Day 1 to Day 85)

Starting on day 1, patients were randomized to receive either REGN1033or matching placebo. The doses were as follows:

300 mg SC Q2W for a total of 6 treatments

300 mg SC Q4W for a total of 3 treatments (with placebo on alternatingweeks to maintain the blind)

100 mg SC Q4W for a total of 3 treatments (with placebo on alternatingweeks to maintain the blind)

Matching placebo SC Q2W for a total of 6 treatments

The injections were administered in the abdomen. Patients were observedfor 30 minutes for vital signs and collection of adverse events (AEs),including occurrence of injection site reactions. Efficacy and safetyprocedures were performed, as well as patient-reported outcomes (PROs).Blood samples were collected for pharmacokinetics (PK), anti-drugantibodies (ADAs), and research. All blood samples were collected afteran overnight fast and before dosing.

Follow-Up (Day 86 to Day 141)

Follow-up visits were on day 141, 8 weeks after the end of treatmentvisit on day 85.

Endpoints

The primary endpoint in the study was the percent change in total leanbody mass measured by DEXA from baseline to week 12. The secondaryendpoints were: TEAEs from baseline to the end of the study, changesfrom baseline in Appendicular lean mass by DEXA, maximal leg pressstrength, 1-repetition max (1-RM), maximal chest press strength (1-RM),4M gait speed, SPPB and SPPB subscores, distance walked in the 6MWT,regional and total fat mass by DEXA, and hand grip strength by handhelddynamometer.

Procedures and Assessments

Safety and tolerability of REGN1033 were assessed by vital signs,electrocardiogram (ECG), echocardiogram, Adverse events (AEs), andclinical laboratory evaluations. Patients were asked to monitor andreport all AEs experienced from the time the informed consent is signeduntil the end of study visit.

Efficacy was assessed by DEXA, strength measures (leg press, chestpress, and handgrip strength), and function measures (stair climb, SPPB,4 Mgait speed, and 6MWT). Other measures used were accelerometry andPROs (the 10-item Physical Function Form [PF-10], the FunctionalAssessment of Chronic Illness Therapy[FACIT] Fatigue Scale, the HealthAssessment Questionnaire Disability Index [HAQ-DI], the Mini-NutritionalAssessment short form [MNA-SF], and the Rapid Assessment of PhysicalActivity [RAPA] questionnaire).

Results

Ninety-five % of randomized patients completed the study. As shown belowin Table 4, at each of the three dose regimens tested, REGN1033treatment significantly increased total lean body mass from baseline toweek 12 compared with placebo; mean differences from placebo were 1.7%(p=0.008), 1.8% (p=0.004) and 2.3% (p<0.001) for REGN1033 100 mg SC Q4W,300 mg Q4W, and 300 mg Q2W respectively, corresponding to lean massincreases of 0.7, 0.8 and 1.0 kg. Appendicular lean mass alsosignificantly increased in patients treated with REGN1033:placebo-adjusted changes ranged from 2.3-2.8%. DEXA-measured total fatmass, android fat mass, and gynoid fat mass all showed numericaldecreases with REGN1033 treatment. REGN1033 treatment resulted indirectionally greater mean changes from baseline in various measures ofstrength and function relative to placebo.

TABLE 4 REGN 100 mg Q4W REGN 300 mg Q4W REGN 300 mg Q2W (n = 62) vs. PBO(n = 65) (n = 64) vs. PBO (n = 65) (n = 59) vs. PBO (n = 65) PrimaryEfficacy Endpoint: DEXA - Percent change from baseline to Week 12Percent change in total lean 1.191% vs. −0.474%  1.308% vs. −0.474% 1.816% vs.−0.474% mass by dual energy x-ray  d = 1.664% (p = 0.0077)  d= 1.781% (p = 0.0043)  d = 2.289% (p = 0.0004) absorptiometry (DEXA)from baseline Summary of Secondary Efficacy: DEXA - Percent change frombaseline to Week 12 Percent change in 2.162% vs. −0.249%  2.033% vs.−0.249%  2.502% vs. −0.249% Appendicular lean mass (g)  d = 2.412% (p =0.0026)  d = 2.282% (p = 0.0043)  d = 2.751% (p = 0.0008) by DEXAPercent change in Total fat −0.077% vs. −0.076%  −2.666% vs. −0.076%−0.947% vs. −0.076% mass (g) determined by d = −0.001% (p = 0.9993) d =−2.590% (p = 0.0099) d = −0.871% (p = 0.3945) DEXA Percent change inAndroid −0.496% vs. 2.501%  −3.164% vs. 2.501%  −2.209% vs. 2.501%  fatmass (g) by DEXA d = −2.998% (p = 0.0587) d = −5.665% (p = 0.0004) d =−4.710% (p = 0.0038) Percent change from 0.022% vs. 0.156%  −2.608% vs.0.156%  −2.185% vs. 0.156%  baseline in Gynoid fat d = −0.135% (p =0.9050) d = −2.751% (p = 0.0152) d = −2.341% (p = 0.0434) mass (g) byDEXA

REGN1033 was generally safe and well tolerated. The frequency of adverseevents was similar across treatment groups. The percentage of subjectsexperiencing at least one SAE was also similar across all treatmentgroups (7.7% in placebo group vs. 7.4% in REGN1033-treated groups).There was no discernable pattern to the distribution of SAEs. There wereno clinically significant trends observed for laboratory tests, vitalsigns, ECGs and echocardiograms.

Conclusions

REGN1033 treatment significantly increased total lean and appendicularlean mass in patients with sarcopenia and was well tolerated.

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described herein will become apparent to those skilledin the art from the foregoing description and the accompanying figures.Such modifications are intended to fall within the scope of the appendedclaims. All publications referred to herein are hereby incorporated byreference.

1. A method for increasing lean body mass in a subject experiencing orat risk of age-related loss of lean muscle mass and/or a disease,disorder, or condition associated with a decrease in skeletal musclemass comprising: a. providing an exercise regimen for the subject; andb. administering a composition comprising an effective amount of aGrowth and Differentiation Factor-8 (GDF-8) inhibitor.
 2. The method ofclaim 1, wherein the effective amount is at least 400 mg.
 3. The methodof claim 1, wherein the effective amount comprises a dosing regimenselected from the group consisting of at least about 0.01 mg/kg to about20 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5mg/kg, about 1 mg/kg to about 10 mg/kg, and about 10 mg/kg to about 100mg/kg.
 4. (canceled)
 5. The method of claim 1, wherein the exerciseregimen comprises resistance training.
 6. The method of claim 5, whereinthe resistance training includes a set of exercises for training of amuscle selected from the group consisting of the dominant hand,non-dominant hand, leg, arm, chest and combinations thereof.
 7. Themethod of claim 1, wherein the composition is administered at least onceevery six weeks, once every five weeks, once every four weeks, onceevery three weeks, once every two weeks, once a week, twice a week,three times a week, four times a week, or five times a week.
 8. Themethod of claim 1, wherein the composition is formulated forintravenous, subcutaneous, intramuscular, or oral administration.
 9. Themethod of claim 8, wherein the composition is formulated forsubcutaneous delivery.
 10. The method of claim 1, wherein the GDF-8inhibitor is an antibody or antigen-binding fragment thereof thatspecifically binds GDF-8.
 11. The method of claim 10, wherein theantibody or antigen-binding fragment thereof comprises the heavy chaincomplementarity determining regions (HCDRs) contained within a heavychain variable region (HCVR) amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 34, SEQ ID NO: 50,SEQ ID NO: 66, SEQ ID NO: 82, SEQ ID NO: 98, SEQ ID NO: 114, SEQ ID NO:130, SEQ ID NO: 146, SEQ ID NO: 162, SEQ ID NO: 178, SEQ ID NO: 194, SEQID NO: 210, SEQ ID NO: 226, SEQ ID NO: 242, SEQ ID NO: 258, SEQ ID NO:274, SEQ ID NO: 290, SEQ ID NO: 306, SEQ ID NO: 360, and SEQ ID NO: 376,or a substantially identical amino acid sequence thereof.
 12. The methodof claim 11, wherein the antibody or antigen-binding fragment thereofcomprises the light chain complementarity determining regions (LCDRs)contained within a light chain variable region (LCVR) amino acidsequence selected from the group consisting of SEQ ID NO:10, SEQ IDNO:26, SEQ ID NO:42, SEQ ID NO:58, SEQ ID NO:74, SEQ ID NO:90, SEQ IDNO:106, SEQ ID NO:122, SEQ ID NO:138, SEQ ID NO:154, SEQ ID NO:170, SEQID NO:186, SEQ ID NO:202, SEQ ID NO:218, SEQ ID NO:234, SEQ ID NO:250,SEQ ID NO:266, SEQ ID NO:282, SEQ ID NO:298, SEQ ID NO:314, SEQ IDNO:322, SEQ ID NO:368, and SEQ ID NO:384, or a substantially identicalamino acid sequence thereof.
 13. The method of claim 10, wherein theantibody or antigen-binding fragment thereof comprises a HCVR amino acidsequence and a LCVR amino acid sequence, wherein the HCVR/LCVR sequencepair is selected from the group consisting of SEQ ID NO: 2/10, SEQ IDNO: 18/26, SEQ ID NO: 34/42, SEQ ID NO: 50/58, SEQ ID NO: 66/74, SEQ IDNO: 82/90, SEQ ID NO: 98/106, SEQ ID NO: 114/122, SEQ ID NO: 130/138,SEQ ID NO: 146/154, SEQ ID NO: 162/170, SEQ ID NO: 178/186, SEQ ID NO:194/202, SEQ ID NO: 210/218, SEQ ID NO: 226/234, SEQ ID NO: 242/250, SEQID NO: 258/266, SEQ ID NO: 274/282, SEQ ID NO: 290/298, SEQ ID NO:306/314, SEQ ID NO: 114/322, SEQ ID NO: 360/368, and SEQ ID NO: 376/384.14. The method of claim 10, wherein the antibody or antigen-bindingfragment thereof binds to an epitope of mature GDF-8 protein comprisingthe amino acid sequence of SEQ ID NO: 340 within amino acids residuesselected from the group consisting of 1 to 109; 1 to 54; 1 to 44; 1 to34; 1 to 24; and 1 to 14; 65 to 72; 35 to 109; 45 to 109; 55 to 109; 65to 109; 75 to 109; 85 to 109; 92 to 109; 95 to 109; 48 to 72; 48 to 69;48 to 65; 52 to 72; 52 to 65; and 56 to
 65. 15.-16. (canceled)
 17. Themethod of claim 10, wherein the antibody or antigen-binding fragmentthereof is a multispecific antibody or antigen-binding fragment thereofthat may bind to 2 or more epitopes of GDF-8.
 18. The method of claim10, wherein the antibody or antigen-binding fragment thereof comprisesthe HCDR3 amino acid sequence selected from the group consisting of SEQID NO: 8, SEQ ID NO: 24, SEQ ID NO: 40, SEQ ID NO: 56, SEQ ID NO: 72,SEQ ID NO: 88, SEQ ID NO: 104, SEQ ID NO: 120, SEQ ID NO: 136, SEQ IDNO: 152, SEQ ID NO: 168, SEQ ID NO: 184, SEQ ID NO: 200, SEQ ID NO: 216,SEQ ID NO: 232, SEQ ID NO: 248, SEQ ID NO: 264, SEQ ID NO: 280, SEQ IDNO: 296, SEQ ID NO: 312, SEQ ID NO: 366, and SEQ ID NO: 382, or asubstantially identical sequence thereof.
 19. The method of claim 18,wherein the antibody or antigen-binding fragment thereof comprises theLCDR3 amino acid sequence selected from the group consisting of SEQ IDNO:16, SEQ ID NO: 32, SEQ ID NO: 48, SEQ ID NO: 64, SEQ ID NO: 80, SEQID NO: 96, SEQ ID NO: 112, SEQ ID NO: 128, SEQ ID NO: 144, SEQ ID NO:160, SEQ ID NO: 176, SEQ ID NO: 192, SEQ ID NO: 208, SEQ ID NO: 224, SEQID NO: 240, SEQ ID NO: 256, SEQ ID NO: 272, SEQ ID NO: 288, SEQ ID NO:304, SEQ ID NO: 320, SEQ ID NO: 328, SEQ ID NO: 374, and SEQ ID NO: 390,or a substantially identical sequence thereof.
 20. The method of claim19, wherein the antibody or antigen-binding fragment thereof comprisesthe HCDR3/LCDR3 amino acid sequence pair selected from the groupconsisting of SEQ ID NO: 8/16, SEQ ID NO: 24/32, SEQ ID NO: 40/48, SEQID NO: 56/64, SEQ ID NO: 72/80, SEQ ID NO: 88/96, SEQ ID NO: 104/112,SEQ ID NO: 120/128, SEQ ID NO: 136/144, SEQ ID NO: 152/160, SEQ ID NO:168/176, SEQ ID NO: 184/192, SEQ ID NO: 200/208, SEQ ID NO: 216/224, SEQID NO: 232/240, SEQ ID NO: 248/256, SEQ ID NO: 264/272, SEQ ID NO:280/288, SEQ ID NO: 296/304, SEQ ID NO: 312/320, SEQ ID NO: 120/328, SEQID NO: 366/374, and SEQ ID NO: 382/390.
 21. The method of any of claim20, wherein the antibody or antigen-binding fragment thereof comprisesthe HCDR1 amino acid sequence selected from the group consisting of SEQID NO: 4, SEQ ID NO: 20, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 68,SEQ ID NO: 84, SEQ ID NO: 100, SEQ ID NO: 116, SEQ ID NO: 132, SEQ IDNO: 148, SEQ ID NO: 164, SEQ ID NO: 180, SEQ ID NO: 196, SEQ ID NO: 212,SEQ ID NO: 228, SEQ ID NO: 244, SEQ ID NO: 260, SEQ ID NO: 276, SEQ IDNO: 292, SEQ ID NO: 308, SEQ ID NO: 362, and SEQ ID NO: 378, or asubstantially identical sequence thereof.
 22. The method of claim 21,wherein the antibody or antigen-binding fragment thereof comprises theHCDR2 amino acid sequence selected from the group consisting of SEQ IDNO: 6, SEQ ID NO: 22, SEQ ID NO: 38, SEQ ID NO: 54, SEQ ID NO: 70, SEQID NO: 86, SEQ ID NO: 102, SEQ ID NO: 118, SEQ ID NO: 134, SEQ ID NO:150, SEQ ID NO: 166, SEQ ID NO: 182, SEQ ID NO: 198, SEQ ID NO: 214, SEQID NO: 230, SEQ ID NO: 246, SEQ ID NO: 262, SEQ ID NO: 278, SEQ ID NO:294, SEQ ID NO: 310, SEQ ID NO: 364, and SEQ ID NO: 380, or asubstantially identical sequence thereof.
 23. The method of claim 22,wherein the antibody or antigen-binding fragment thereof furthercomprises the LCDR1 amino acid sequence selected from the groupconsisting of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO:60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ IDNO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, SEQ ID NO: 204,SEQ ID NO: 220, SEQ ID NO: 236, SEQ ID NO: 252, SEQ ID NO: 268, SEQ IDNO: 284, SEQ ID NO: 300, SEQ ID NO: 316, SEQ ID NO: 324, SEQ ID NO: 370,and SEQ ID NO: 386 or a substantially identical sequence thereof. 24.The method of claim 23, wherein the antibody or antigen-binding fragmentthereof further comprises the LCDR2 amino acid sequence selected fromthe group consisting of SEQ ID NO: 14, SEQ ID NO: 30, SEQ ID NO: 46, SEQID NO: 62, SEQ ID NO: 78, SEQ ID NO: 94, SEQ ID NO: 110, SEQ ID NO: 126,SEQ ID NO: 142, SEQ ID NO: 158, SEQ ID NO: 174, SEQ ID NO: 190, SEQ IDNO: 206, SEQ ID NO: 222, SEQ ID NO: 238, SEQ ID NO: 254, SEQ ID NO: 270,SEQ ID NO: 286, SEQ ID NO: 302, SEQ ID NO: 318, SEQ ID NO: 326, SEQ IDNO: 372, and SEQ ID NO: 388 or a substantially identical sequencethereof.
 25. The method of claim 10, wherein the antibody orantigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 aminoacid sequences selected from the group consisting of SEQ ID NO:36/38/40, SEQ ID NO: 116/118/120, SEQ ID NO: 228/230/232, SEQ ID NO:362/364/366, and SEQ ID NO: 378/380/382.
 26. The method of claim 25,wherein the antibody or antigen-binding fragment thereof comprisesLCDR1, LCDR2 and LCDR3 amino acid sequences selected from the groupconsisting of SEQ ID NO: 44/46/48, SEQ ID NO: 124/126/128, SEQ ID NO:236/238/240, SEQ ID NO: 370/372/374, and SEQ ID NO: 386/388/390.
 27. Themethod of claim 26, wherein the heavy and light chain CDRs comprise theamino acid sequences selected from the group consisting of SEQ ID NO:36/38/40/44/46/48, SEQ ID NO: 116/118/120/124/126/128, SEQ ID NO:228/230/232/236/238/240, SEQ ID NO: 362/364/366/370/372/374, and SEQ IDNO: 378/380/382/386/388/390, respectively.
 28. The method of claim 10,wherein the antibody or antigen-binding fragment thereof comprises theheavy and light chain CDR domains contained within heavy and light chainvariable reions (HCVR/LCVR) comprising the amino acid sequence pairsselected from the group consisting of SEQ ID NO: 2/10, SEQ ID NO: 18/26,SEQ ID NO: 34/42, SEQ ID NO: 50/58, SEQ ID NO: 66/74, SEQ ID NO: 82/90,SEQ ID NO: 98/106, SEQ ID NO: 114/122, SEQ ID NO: 130/138, SEQ ID NO:146/154, SEQ ID NO: 162/170, SEQ ID NO: 178/186, SEQ ID NO: 194/202, SEQID NO: 210/218, SEQ ID NO: 226/234, SEQ ID NO: 242/250, SEQ ID NO:258/266, SEQ ID NO: 274/282, SEQ ID NO: 290/298, SEQ ID NO: 306/314, SEQID NO: 114/322, SEQ ID NO: 360/368, and SEQ ID NO: 376/384.
 29. Themethod of claim 10, wherein the antibody or antigen-binding fragmentthereof comprises the HCDRs contained within the HCVR encoded by thenucleotide sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 17, SEQ ID NO: 33, SEQ ID NO: 49, SEQ ID NO: 65, SEQ ID NO:81, SEQ ID NO: 97, SEQ ID NO: 113, SEQ ID NO: 129, SEQ ID NO: 145, SEQID NO: 161, SEQ ID NO: 177, SEQ ID NO: 193, SEQ ID NO: 209, SEQ ID NO:225, SEQ ID NO: 241, SEQ ID NO: 257, SEQ ID NO: 273, SEQ ID NO: 289, SEQID NO: 305, SEQ ID NO: 359, and SEQ ID NO: 375, or a substantiallysimilar sequence having at least 95% homology thereof.
 30. The method ofclaim 29, wherein the antibody or antigen-binding fragment thereofcomprises the LCDRs contained within the LCVR encoded by the nucleotidesequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO:25, SEQ ID NO: 41, SEQ ID NO: 57, SEQ ID NO: 73, SEQ ID NO: 89, SEQ IDNO: 105, SEQ ID NO: 121, SEQ ID NO: 137, SEQ ID NO: 153, SEQ ID NO: 169,SEQ ID NO: 185, SEQ ID NO: 201, SEQ ID NO: 217, SEQ ID NO: 233, SEQ IDNO: 249, SEQ ID NO: 265, SEQ ID NO: 281, SEQ ID NO: 297, SEQ ID NO: 313,SEQ ID NO: 321, SEQ ID NO: 367, and SEQ ID NO: 383 or a substantiallysimilar sequence having at least 95% homology thereof. 31.-84.(canceled)
 85. The method of claim 1, wherein the subject isexperiencing age related loss of lean muscle mass and/or post-surgicalmuscle wasting.
 86. The method of claim 1, wherein the subject is 60years of age or older.
 87. The method of claim 1, wherein the subject isa human subject. 88.-92. (canceled)
 93. The method of claim 1, whereinthe disease, disorder, or condition is selected from the groupconsisting of sarcopenia, cachexia, muscle injury, muscle wasting,muscle atrophy, metabolic condition, and sporadic inclusion bodymyositis (sIBM).
 94. The method of claim 93, wherein the sarcopenia isassociated with aging.
 95. The method of claim 93, wherein the cachexiais idiopathic or secondary to other conditions.
 96. The method of claim95, wherein the other conditions are selected from the group consistingof cancer, chronic renal failure, diabetes, or chronic obstructivepulmonary disease.
 97. The method of claim 93, wherein the muscleatrophy or muscle wasting is caused by or associated with disuse,limited mobility, immobilization, bed rest, injury, medical treatment,surgical intervention, or mechanical ventilation.
 98. The method ofclaim 97, wherein the limited mobility is associated with recentmyocardial infarction, unstable cardiac conditions, acute heart failure,severe myocarditis, uncontrolled hypertension, cardiac valve diseaserequiring surgery, and severe aortic stenosis.
 99. The method of claim97, wherein the surgical intervention is orthopedic surgery.
 100. Themethod of claim 99, wherein the orthopedic surgery is selected from thegroup consisting of hip replacement, hip fracture surgery, and kneereplacement.
 101. The method of claim 5, wherein the resistance trainingcomprises weight training, use of resistance band, use of exercisemachine, and/or use of the subject's own body weight.
 102. The method ofclaim 1, wherein the increase in lean body mass is determined by dualx-ray absorptiometry compared to baseline.
 103. The method of claim 102,wherein the increase in lean body mass is an increase in total lean bodymass determined by dual x-ray absorptiometry compared to baseline. 104.The method of claim 1, further comprising administering an additionaltherapeutically active component.
 105. The method of claim 104, whereinthe additional therapeutically active component is selected from thegroup consisting of an additional GDF8 inhibitor, growth factorinhibitors, immunosuppressants, anti-inflammatory agents, metabolicinhibitors, enzyme inhibitors, and cytotoxic/cytostatic agents.
 106. Themethod of claim 105, wherein the additional GDF-8 inhibitor is a smallmolecule inhibitor of GDF-8, additional GDF-8 antibody, or GDF-8 bindingmolecule.
 107. The method of claim 104, wherein the additionaltherapeutically active component is administered prior to, concurrentwith, or after the administration of the GDF-8 inhibitor.
 108. Themethod of claim 1, wherein the disease, disorder, or condition isassociated with GDF-8 activity.